WO2019182037A1 - 毒性が低減されたアンチセンスオリゴヌクレオチド - Google Patents
毒性が低減されたアンチセンスオリゴヌクレオチド Download PDFInfo
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- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7125—Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
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- 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
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- C12N2310/32—Chemical structure of the sugar
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- C12N2310/00—Structure or type of the nucleic acid
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- C12N2310/34—Spatial arrangement of the modifications
- C12N2310/341—Gapmers, i.e. of the type ===---===
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- C12N2320/53—Methods for regulating/modulating their activity reducing unwanted side-effects
Definitions
- the present invention relates to an antisense oligonucleotide with reduced toxicity.
- Nucleic acid drugs are drugs made of nucleic acids (oligonucleotides) that form complementary base pairs with target DNA or RNA, and are expected as new drugs.
- Various artificial nucleic acid units artificial nucleotides which are artificial nucleosides or phosphate adducts thereof) have been developed as nucleic acid units used in nucleic acid medicines. For example, it is known that affinity to a target nucleic acid and resistance to a nuclease are improved by converting the oxygen atom at the 2′-position of the ribonucleotide to methoxyethyl (MOE) (for example, patent documents) 1).
- MOE methoxyethyl
- 2 ′, 4′-BNA and 2 ′, 4′-LNA are compounds in which the 2′-position and 4′-position of the sugar moiety of the nucleic acid unit are cross-linked, and are known to have high affinity for the target nucleic acid.
- nucleotides in which the 2′-position and the 3′-position are crosslinked (2′-3′-bridged nucleotides), and nucleotides in which alkyl is introduced at the ⁇ -position of the sugar part 3′-position (3′-modified non-crosslinked) Nucleotides) are also known.
- the influence of RNase H on RNA strand cleavage activity by introducing these artificial nucleic acids into a DNA strand has been studied (for example, see Non-Patent Documents 1 and 2).
- Gapmer-type antisense nucleic acid forms a double-stranded complex with target RNA, and intracellular RNase H is known to recognize the double-stranded part of deoxyribonucleotide part and target RNA and cleave the RNA chain. ing.
- RNase H intracellular RNase H is known to recognize the double-stranded part of deoxyribonucleotide part and target RNA and cleave the RNA chain.
- high sequence specificity is required.
- toxicity due to the off-target effect has been reported (for example, see Non-Patent Documents 3 and 4).
- the off-target effect occurs when an antisense nucleic acid and an RNA having a similar sequence other than the target form a double-stranded complex, and the RNA other than the target is cleaved.
- modification methods that reduce such toxicity.
- SNP single nucleotide polymorphism
- An object of the present invention is to provide an antisense oligonucleotide with reduced toxicity.
- the present inventors have made nucleotides bridging the 2′-position and 3′-position of the sugar moiety (2′-3′-bridged nucleotide) and / or non-bridged nucleotides having a substituent at the 3′-position (modification at the 3′-position).
- the present inventors have found that a gapmer-type antisense nucleic acid having a non-crosslinked nucleotide in the central region has low toxicity and high sequence selectivity.
- the present invention includes the following aspects.
- the central region consists of 5-15 nucleotides;
- the 5 ′ region and the 3 ′ region are each independently composed of 1 to 7 nucleotides.
- the central region consists of 8-12 nucleotides;
- the 5 ′ region and the 3 ′ region are each independently composed of 2 to 5 nucleotides.
- the 3′-modified non-bridging nucleotide contained in the central region is Formula (II) below: (Where Bx is the nucleobase moiety; X is O or S; R 12 is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyl substituted with one or more substituents, C2-C6 alkenyl substituted with one or more substituents, C2-C6 alkynyl substituted with one or more substituents, acyl, acyl substituted with one or more substituents, amide substituted with one or more substituents, hydroxy, C1-C6 alkoxy, one or more A C1-C6 alkoxy, sulfanyl, C1-C6 alkylthio substituted with one or more substituents of C1-C6 alkylthio substituted with one or more substituents; wherein each of the substituents is independently halogen atom, o
- —Q 1 — is —O—, —NH—, —NR 6 — or —S—, R 6 is C1-C12 alkyl, and —Q 2 — is —CH 2 —.
- R 1 , R 2 and R 3 are hydrogen atoms
- R 1 , R 2 and R 3 are hydrogen atoms
- X is O.
- To 9. The antisense oligonucleotide according to any one of the above.
- the central region is a gap region;
- the 5 ′ side region is a 5 ′ wing region;
- the 3 ′ side region is a 3 ′ wing region;
- To 10. The antisense oligonucleotide according to any one of the above.
- the sugar moiety-modified nucleotides contained in the 5′-side region and the 3′-side region are each independently selected from the group consisting of a 2′-modified non-crosslinked nucleotide and 2 ′, 4′-BNA.
- the antisense oligonucleotide according to any one of the above.
- the 2′-modified non-crosslinked nucleotide is a 2′-O-methylated nucleotide, 2′-O-methoxyethyl (MOE) nucleotide, 2′-O-aminopropyl (AP) nucleotide, 2′-fluorinated 11. at least one selected from the group consisting of nucleotides, 2′-O- (N-methylacetamido) (NMA) ated nucleotides and 2′-O-methylcarbamoylethyl (MCE) ated nucleotides; An antisense oligonucleotide according to 1.
- the 2 ′, 4′-BNA is at least one selected from the group consisting of LNA, cEt-BNA, ENA, BNA NC , AmNA, and scpBNA.
- the antisense oligonucleotide contains a phosphorothioate linkage; To 14. The antisense oligonucleotide according to any one of the above.
- the functional molecule is selected from the group consisting of sugars, lipids, peptides and proteins and derivatives thereof; 16.
- the functional molecule is a lipid selected from the group consisting of cholesterol, tocopherol and tocotrienol, 16. Or 17.
- the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor, Or 17.
- the functional molecule is a peptide or protein selected from the group consisting of a receptor ligand and an antibody; Or 17.
- An oligonucleotide in which an oligonucleotide chain comprising at least one ribonucleotide is linked to a group derived from the antisense oligonucleotide according to any one of (1) and (iv) at least four consecutive sequences recognized by RNaseH An oligonucleotide complex comprising an oligonucleotide comprising an oligonucleotide chain comprising a nucleotide comprising: An oligonucleotide complex in which the oligonucleotide chain comprising at least one ribonucleotide of (iii) and the oligonucleotide chain comprising at least 4 consecutive nucleotides recognized by RNaseH of (iv) hybridize .
- a method for controlling the function of a target RNA in a mammal comprising a step of administering the pharmaceutical composition described in 1. to the mammal.
- a method for controlling the expression of a target gene in a mammal comprising a step of administering the pharmaceutical composition described in 1. to the mammal.
- nucleotide selected from the group consisting of a 2'-3 'bridged nucleotide and a 3' modified non-bridged nucleotide To 20. 20. The antisense oligonucleotide according to any one of 21. A method for producing the prodrug described in 1.
- the present invention provides an antisense oligonucleotide with reduced toxicity.
- Example 2 is a graph showing the effect of the antisense oligonucleotide according to the present embodiment (Example 1) on the expression level of SOD-1 in mouse brain endothelial cells. It is a graph which shows the influence on the cell survival rate in the mouse
- N- means normal, “i-” means iso, “s-” means secondary, “t-” means tertiary, “m-” means meta, and “p-” means para.
- Ph means phenyl
- Me means methyl
- Pr means propyl
- Bu means butyl
- DMTr means dimethoxytrityl.
- the functional group substituted with a protecting group means a functional group in which a hydrogen atom of the functional group is substituted with a protecting group.
- Halogen atom means a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.
- C1-C12 alkyl means a monovalent group of a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 12 carbon atoms.
- Examples of C1-C12 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, n-heptyl, Examples include n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl and the like.
- C1-C6 alkyl means a monovalent group of a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 6 carbon atoms in the “C1-C12 alkyl”.
- Examples of C1-C6 alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl and the like.
- C1-C3 alkyl means a monovalent group of a straight-chain or branched saturated aliphatic hydrocarbon having 1 to 3 carbon atoms.
- Halo C1-C6 alkyl means a group in which at least one hydrogen atom at any position of the “C1-C6 alkyl” is substituted with the “halogen atom”.
- C2-C6 alkenyl means a monovalent group of a straight-chain or branched unsaturated aliphatic hydrocarbon having 2 to 6 carbon atoms containing at least one carbon-carbon double bond.
- Examples of C2-C6 alkenyl include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, butadienyl, 3-methyl-2-butenyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl and the like.
- C2-C6 alkynyl means a monovalent group of a linear or branched unsaturated aliphatic hydrocarbon having 2 to 6 carbon atoms and containing at least one carbon-carbon triple bond.
- Examples of C2-C6 alkynyl include ethynyl, propargyl, 3-butynyl, 4-pentynyl and the like.
- acyl means a group in which a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl or aryl is bonded to a carbonyl (—C ( ⁇ O) —) group.
- acyl include formyl, acetyl, pivaloyl, benzoyl and the like.
- haloacyl means a group in which at least one hydrogen atom at any position of the “acyl” is substituted with the “halogen atom”.
- “Amido” is an aminocarbonyl (—CONH 2 ) group or a group in which at least one hydrogen atom of the aminocarbonyl group is independently selected from the group consisting of C1-C6 alkyl, C2-C6 alkenyl and aryl. Means a substituted group.
- the amide include carbamoyl, methylaminocarbonyl, isopropylaminocarbonyl, phenylaminocarbonyl and the like.
- C1-C6 alkoxy means a group in which the “C1-C6 alkyl” is bonded to an oxy (—O—) group.
- Examples of C1-C6 alkoxy include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, n-hexyloxy and the like. It is done.
- C1-C6 alkylthio means a group in which the “C1-C6 alkyl” is bonded to a thio (—S—) group.
- Examples of C1-C6 alkylthio include methylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio, isobutylthio, s-butylthio, t-butylthio, n-pentylthio, isopentylthio, n-hexylthio and the like.
- C2-C50 alkylene means a divalent group of a straight or branched saturated aliphatic hydrocarbon having 2 to 50 carbon atoms.
- C2-C20 alkylene means a divalent group of a straight or branched saturated aliphatic hydrocarbon having 2 to 20 carbon atoms.
- C8-C12 alkylene means a divalent group of a straight-chain or branched saturated aliphatic hydrocarbon having 8 to 12 carbon atoms in the “C2-C20 alkylene”.
- C2-C6 alkylene means a divalent group of a straight-chain or branched saturated aliphatic hydrocarbon having 2 to 6 carbon atoms in the “C2-C20 alkylene”.
- Ethylene ethanediyl
- propylene propane-1,3-diyl (trimethylene), propane-2,2-diyl (isopropylidene), 2,2-dimethyl-propane-1,3-diyl, hexane-1,6- Examples include diyl (hexamethylene) and 3-methylbutane-1,2-diyl.
- C2-C20 alkenylene means a divalent group of a linear or branched unsaturated aliphatic hydrocarbon having 2 to 20 carbon atoms and containing at least one carbon-carbon double bond. To do.
- “Mono C1-C6 alkylamino” means a group in which one hydrogen atom of an amino (NH 2 ) group is replaced by the above “C1-C6 alkyl”, and examples thereof include methylamino, ethylamino, n- Examples include propylamino, isopropylamino, n-butylamino, isobutylamino, s-butylamino, t-butylamino, n-pentylamino, n-hexylamino and isohexylamino.
- “DiC1-C6 alkylamino” means a group in which two of the hydrogen atoms of an amino (NH 2 ) group are replaced with the same or different two “C1-C6 alkyl”, for example, dimethylamino, Examples include diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, di-n-pentylamino, di-n-hexylamino, N-methyl-N-ethylamino, and N-methyl-N-isopropylamino. .
- C1-C6 alkylcarbonyl “halo C1-C6 alkylcarbonyl”, “C1-C6 alkoxycarbonyl”, “mono-C1-C6 alkylaminocarbonyl” and “di-C1-C6 alkylaminocarbonyl” are “C1-C6 alkyl”, “halo C1-C6 alkyl”, “C1-C6 alkoxy”, “mono-C1-C6 alkylamino” and “di-C1-C6 alkylamino” are carbonyl (—C ( ⁇ O) —) It means a group bonded to a group.
- C1-C6 alkylsulfonyl “halo C1-C6 alkylsulfonyl”, “C1-C6 alkoxysulfonyl”, “mono-C1-C6 alkylaminosulfonyl” and “di-C1-C6 alkylaminosulfonyl” are “C1-C6 alkyl”, “halo C1-C6 alkyl”, “C1-C6 alkoxy”, “mono-C1-C6 alkylamino” and “di-C1-C6 alkylamino” are sulfonyl groups (—S (O) 2 — ) Means a group bonded to.
- the “ribonucleoside group” means a group in which a base is bonded to the carbon atom at the 1′-position of ribose and the 3′-position and 5′-position hydroxy groups of the ribose are removed.
- the base moiety in the ribonucleoside group in the present invention may be a naturally occurring base or a base obtained by modifying a naturally occurring base.
- the base moiety may be modified in combination of a plurality of types for one ribonucleoside group. Examples of such modifications include Journal of Medicinal Chemistry (2016, 59, 21, 9645-9667), Medicinal Chemistry Communications (2014, 5, 1454-1471), Future Medicinal. -It is described in Chemistry (2011, Vol. 3, No. 3, 339-365).
- Deoxyribonucleoside group means a group in which a base is bonded to the 1′-position carbon atom of 2′-deoxyribose and the 3′-position and 5′-position hydroxy groups of the 2′-deoxyribose are removed.
- the base moiety in the deoxyribonucleoside group in the present invention may be a naturally occurring base or a base obtained by modifying a naturally occurring base.
- the base moiety may be modified in combination of a plurality of types for one deoxyribonucleoside group. Examples of such modifications include Journal of Medicinal Chemistry (2016, 59, 21, 9645-9667), Medicinal Chemistry Communications (2014, 5, 1454-1471), Future Medicinal. Chemistry (2011, Vol. 3, No. 3, pp. 339-365).
- Oxo refers to a group ( ⁇ O) in which an oxygen atom is substituted via a double bond. When oxo is substituted with a carbon atom, it forms a carbonyl together with the carbon atom.
- Thioxo refers to a group ( ⁇ S) in which an oxygen atom is substituted via a double bond. When thioxo is substituted with a carbon atom, it forms a thiocarbonyl together with the carbon atom.
- the hydroxy protecting group and the amino protecting group are not particularly limited as long as they are stable when synthesizing an antisense oligonucleotide.
- Protective Group In Organic Synthesis 4th edition, T well known to those skilled in the art.
- Protecting groups described in W. Greene, P. G. M. Wuts, John Wiley & Sons Inc. (2006) and the like can be mentioned.
- amino protecting group includes acyl (eg, formyl, acetyl, propionyl, pivaloyl (Pv), tigloyl, etc.), haloacyl (eg, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloro).
- acyl eg, formyl, acetyl, propionyl, pivaloyl (Pv), tigloyl, etc.
- haloacyl eg, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloro
- Amide protecting groups such as acetyl, trichloroacetyl, etc.), arylcarbonyl (for example, benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, etc.); C1-C6 alkoxycarbonyl (For example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl, n-butoxycarbonyl, i-butoxycarbonyl, t-butoxycarbonyl (Boc), t-amyloxycarboni C2 to C6 alkenyloxycarbonyl (for example, vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc) and the like), tri (C1-C3 alkyl) silyl Ethoxycarbonyl (for example, 2- (trimethylsilyl) ethoxycarbonyl (Teoc) and the like),
- Antisense effect means that the function of a target RNA is controlled by hybridization of a target RNA selected corresponding to the target gene with, for example, an oligonucleotide having a sequence complementary to the partial sequence.
- a target RNA selected corresponding to the target gene with, for example, an oligonucleotide having a sequence complementary to the partial sequence.
- Antisense oligonucleotide is an oligonucleotide that produces the antisense effect. Examples thereof include DNA and oligodeoxyribonucleotides, but are not limited thereto, and RNA, oligoribonucleotides, or oligonucleotides designed so that an antisense effect usually occurs may be used. The same applies to antisense nucleic acids.
- Target RNA means mRNA, mRNA precursor or ncRNA, mRNA transcribed from genomic DNA encoding the target gene, mRNA not subjected to base modification, mRNA precursor not subjected to splicing, ncRNA, etc. including.
- the “target RNA” whose function is controlled by the antisense effect is not particularly limited, and includes RNA associated with a gene whose expression is enhanced in various diseases.
- the “target RNA” may be any RNA synthesized by a DNA-dependent RNA polymerase, preferably mRNA or mRNA precursor. More preferred is mammalian mRNA or mRNA precursor, still more preferred is human mRNA or mRNA precursor, and particularly preferred is human mRNA.
- Hybridization refers to the act of forming a duplex between oligonucleotides containing complementary sequences or groups derived from oligonucleotides, and the groups derived from oligonucleotides or oligonucleotides containing complementary sequences. It means the phenomenon of forming a double chain.
- “Complementary” means that two nucleobases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen-type base pairs, etc.) via hydrogen bonding Means.
- Two oligonucleotides or groups derived from oligonucleotides can “hybridize” if their sequences are complementary.
- oligonucleotides or groups derived from oligonucleotides In order for two oligonucleotides or groups derived from oligonucleotides to hybridize, they need not be completely complementary, but complementarity for two oligonucleotides or groups derived from oligonucleotides to hybridize Is preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, or 99% or more). Sequence complementarity can be determined by utilizing a computer program that automatically identifies subsequences of oligonucleotides. For example, OligoAnalyzer is one such software provided by Integrated DNA Technologies. This program is also available on the website.
- BLAST program based on the nucleotide sequence information of the target RNA.
- BLAST programs see Proceedings of the National Academy of Sciences of the United States of America, 1990, 87, pp 2264-2268, 1993, 90, pp 5873-5877, and Journal of Molecular Biology, 1990, 215, pp -410 etc. can be referenced.
- Nucleotide means a molecule that can be a constituent unit of nucleic acid (oligonucleotide), and usually has a base as a constituent element.
- a nucleotide is composed of, for example, a sugar, a base and a phosphate. Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides described below.
- Oligonucleotide means a molecule having a structure in which one or more nucleotides are polymerized.
- an “oligonucleotide” is composed of one nucleotide, the oligonucleotide can be referred to as a “nucleotide”.
- the nucleotides included in the “antisense oligonucleotide” molecule of the present invention are independently linked to each other by a linking group containing a phosphodiester bond, a modified phosphodiester bond described below, or a non-nucleotide structure described below.
- the 3 ′ terminal nucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at the 3 ′ position, more preferably a hydroxy group, and usually a hydroxy group.
- the 5 ′ terminal nucleotide of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at the 5 ′ position, more preferably has a hydroxy group, and usually has a hydroxy group.
- the “group derived from an oligonucleotide” means a group obtained by removing a hydrogen atom, a hydroxy group, etc. from at least one hydroxy group at the 3 ′ end and 5 ′ end of the oligonucleotide, and other groups (for example, other groups A group derived from an oligonucleotide) and indirectly linked by a covalent bond to form a phosphodiester bond or a modified phosphodiester bond.
- the 3′-terminal or 5′-terminal hydroxy group includes a hydroxy group of a phosphate group.
- Nucleotide sequence means the base sequence of nucleotides constituting an oligonucleotide.
- sequence portion means a partial structure of an oligonucleotide chain.
- sequence portion containing a nucleotide is a partial structure of a region containing the nucleotide in the oligonucleotide chain.
- Deoxyribonucleotide means a molecule in which the sugar is 2′-deoxyribose, a base is bonded to the 1′-position carbon atom of 2′-deoxyribose, and a phosphate group is present at the 3′-position or the 5′-position. To do.
- the deoxyribonucleotide in the present invention may be a naturally-occurring deoxyribonucleotide or a deoxyribonucleotide in which the base portion or phosphodiester-binding portion of a naturally-occurring deoxyribonucleotide is modified.
- Modification of the base moiety and modification of the phosphodiester binding site may be applied to one deoxyribonucleotide in combination.
- Examples of the modified deoxyribonucleotide include Journal ⁇ ⁇ ⁇ of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, etc. It is described in.
- the “deoxyribonucleotide” constitutes the antisense oligonucleotide molecule of the present invention
- the 3′-position of deoxyribonucleotide is a phosphodiester bond or a modified phosphodiester bond (for example, phosphorothioate bond) and other nucleotides etc.
- the 5'-position of deoxyribonucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, phosphorothioate bond).
- the deoxyribonucleotide at the 3 'end of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at the 3' position, and the 5 'position is as described above.
- the deoxyribonucleotide at the 5 'end of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at the 5' position, and the 3 'position is as described above.
- “Oligodeoxyribonucleotide” means an oligonucleotide composed of the deoxyribonucleotide.
- the deoxyribonucleotides constituting the oligodeoxyribonucleotide may be the same or different.
- DNA means an oligonucleotide composed of natural deoxyribonucleotides. Natural deoxyribonucleotides constituting the DNA may be the same or different.
- “Ribonucleotide” means a molecule in which the sugar is ribose, a base is bonded to the 1′-position carbon atom of ribose, and a phosphate group is present at the 2′-position, 3′-position or 5′-position.
- the ribonucleotide in the present invention may be a naturally occurring ribonucleotide or a ribonucleotide in which the base moiety or phosphodiester-binding moiety of a naturally occurring ribonucleotide is modified. Modification of the base moiety and modification of the phosphodiester binding site may be applied to one ribonucleotide in combination.
- modified ribonucleotide examples include JournalJof Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, etc. It is described in.
- the “ribonucleotide” constitutes an antisense oligonucleotide molecule of the present invention
- the 3 ′ position of the ribonucleotide is a phosphodiester bond or a modified phosphodiester bond (eg, a phosphorothioate bond), and the other. It is linked to a nucleotide, and the 5 ′ position of ribonucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (eg, phosphorothioate bond).
- the 3 'terminal ribonucleotide of the antisense oligonucleotide molecule of the present invention preferably has a hydroxy group or a phosphate group at the 3' position, and the 5 'position is as described above.
- the 5 'terminal ribonucleotide of the antisense oligonucleotide molecule preferably has a hydroxy group or a phosphate group at the 5' position, and the 3 'position is as described above.
- “Oligoribonucleotide” means an oligonucleotide composed of the ribonucleotides.
- the ribonucleotides constituting the oligoribonucleotide may be the same or different.
- RNA means an oligonucleotide composed of natural ribonucleotides.
- the natural ribonucleotides constituting the RNA may be the same or different.
- “Sugar-modified nucleotide” means that the sugar moiety of the deoxyribonucleotide or ribonucleotide is partially substituted with one or more substituents, or the entire sugar skeleton is different from ribose and 2′-deoxyribose.
- a sugar skeleton for example, a 5- to 6-membered sugar skeleton such as hexitol, threose, etc.
- the entire sugar skeleton or a ring part of the sugar skeleton is a 5- to 7-membered saturated or unsaturated ring (for example, Cyclohexane, cyclohexene, morpholine, etc.) or a 5- to 7-membered ring is replaced by a partial structure that can be formed by a hydrogen bond (eg, a peptide structure), or the ring of the sugar moiety is opened,
- a ring-opened part is meant a modified nucleotide.
- the base moiety of the “sugar modified nucleotide” may be a naturally occurring base or a modified base.
- the phosphodiester bond portion of the “sugar moiety-modified nucleotide” may be a phosphodiester bond or a modified phosphodiester bond.
- the modification of the base moiety and the modification of the phosphodiester binding site may be performed in combination of a plurality of types on one sugar moiety-modified nucleotide.
- the modification of the ring-opened moiety includes, for example, demethylation in addition to halogenation, alkylation (for example, methylation, ethylation), hydroxylation, amination, and thiolation.
- the “sugar modified nucleotide” may be a crosslinked nucleotide or a non-crosslinked nucleotide.
- sugar-modified nucleotides include Japanese Patent Application Laid-Open No. 10-304889, International Publication No. 2005/021570, Japanese Patent Application Laid-Open No. 10-195098, Japanese Patent Publication No. 2002-521310, International Publication No. 2007/143315, International Public Publication No. 2008/043753, International Publication No. 2008/029619, Journal of Medicinal Chemistry, 2008, 51, p 2766 and International Publication No.
- Antisense Method Literature 2008/049085 (hereinafter referred to as “Antisense Method Literature”) Etc., nucleotides disclosed as suitable for use in antisense methods.
- the documents include hexitol nucleotide (HNA), cyclohexene nucleotide (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threonucleotide (TNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), 2′- O-methylated nucleotide, 2′-O-methoxyethyl (MOE) nucleotide, 2′-O-aminopropyl (AP) nucleotide, 2′-fluorinated nucleotide, 2′-F-arabinonucleotide (2 ′ -F-ANA), bridged nucleotides (BNA (Bridged Nucleic Acid)), 2'-O- (N-
- Nucleotides are disclosed. Furthermore, Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp 2296-2300 (described above, Non-Patent Document 1), The Journal of Biological Chemistry, 2004, 279, pp 36317-36326 (described above, Non-Patent Document 2) Discloses nucleotides such as 2'-3 'bridged nucleotides and 3' modified non-bridged nucleotides. In addition, there are also sugar-modified nucleotides in Journal of Medicinal Chemistry, 2016, 59, pp 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, etc. It is disclosed.
- the “sugar modified nucleotide” constitutes the antisense oligonucleotide molecule of the present invention
- the 3′-position of the sugar modified nucleotide is a phosphodiester bond or a modified phosphodiester bond (eg, phosphorothioate bond). It is linked to other nucleotides and the like, and the 5′-position of the sugar moiety modified nucleotide is linked to another nucleotide or the like by a phosphodiester bond or a modified phosphodiester bond (for example, phosphorothioate bond).
- the sugar-modified nucleotide at the 3 'end of the antisense oligonucleotide molecule of the present invention preferably has, for example, a hydroxy group or a phosphate group at the 3' position, and the 5 'position is as described above.
- the sugar-modified nucleotide at the 5 'end of the antisense oligonucleotide molecule preferably has, for example, a hydroxy group or a phosphate group at the 5' position, and the 3 'position is as described above.
- Examples of modifications of phosphodiester linkages in deoxyribonucleotides, ribonucleotides and sugar modified nucleotides include phosphorothioation, methylphosphonation (including chiral-methylphosphonation), methylthiophosphonation, phosphorodithioation, phosphoro Examples include amidate formation, phosphorodiamidate formation, phosphoramidothioate formation, and boranophosphate formation.
- a “bridged nucleotide” is a sugar-modified nucleotide in which a bridging unit is replaced by two substitutions in the sugar moiety, such as a 2′-4 ′ bridged nucleotide and a 2′-3 ′ bridged nucleotide, 3 ′ -5 'bridged nucleotides and the like.
- a 2′-4 ′ bridged nucleotide (2 ′, 4′-BNA) is a nucleotide having a sugar moiety in which a 2′-position carbon atom and a 4′-position carbon atom are bridged by two or more atoms.
- C2-C6 alkylene (the alkylene is unsubstituted or substituted with one or more substituents selected from the group consisting of halogen atoms, oxo and thioxo, and 1 or 2 of the alkylene)
- Two methylenes are unsubstituted or independently a group consisting of —O—, —NR 13 — (R 13 represents a hydrogen atom, C1-C6 alkyl or haloC1-C6 alkyl) and —S—.
- R 13 is a hydrogen atom, a C1-C6 alkyl or halo C1-C6 alkyl
- - C ( O) -NR 13 -
- R 13 is a hydrogen atom, C1-C6 alkyl or halo C1-C6
- a group represented by —C ( ⁇ S) —NR 13 — R 13 represents a hydrogen atom, C1-C6 alkyl or haloC1-C6 alkyl
- BNA locked nucleic acid (Locked Nucleic Acid (registered trademark)), ⁇ -L-methyleneoxy (4′-CH 2 —O-2 ′) BNA or ⁇ -D— Methyleneoxy (4′-CH 2 —O-2 ′) BNA, ethyleneoxy (4 ′-(CH 2 ) 2 —O-2 ′) BNA, also referred to as ENA, ⁇ -D-thio (4′-CH 2- S-2 ′) BNA, aminooxy (4′-CH 2 —O—N (R 21 ) -2 ′) BNA (R 21 is H or CH 3 ), 2 ′, 4′-BNA Oxyamino (4′-CH 2 —N (R 22 ) —O-2 ′) BNA (R 22 is H or CH 3 ), also referred to as NC , 2 ′, 4′-BNA COC , 3 ′ -Amino-2 ′, 4′-BNA, also referred to as 5′-methyl BNA,
- a 2′-3 ′ bridged nucleotide is a nucleotide having a sugar moiety in which a carbon atom at the 2 ′ position and a carbon atom at the 3 ′ position are bridged by one or more atoms.
- Examples include nucleotides having the partial structure shown (sugar moiety and base moiety).
- the 3'-5 'bridged nucleotide is a nucleotide having a sugar moiety in which the 3'-position carbon atom and the 5'-position carbon atom are bridged by two or more atoms.
- An example is tricyclo-DNA (tcDNA).
- the 3′-modified non-bridged nucleotide is a non-bridged nucleotide in which the 3′-position carbon atom is modified.
- a nucleotide having a partial structure (sugar moiety and base moiety) represented by the following formula (II) can be mentioned.
- Bx is the nucleobase moiety;
- X is O or S;
- a 2′-modified non-bridged nucleotide is a non-bridged nucleotide modified at the 2′-position oxygen atom or carbon atom, for example, 2′-O-methylated nucleotide, 2′-O-methoxyethyl (MOE).
- sugar-modified nucleotides are not limited to those exemplified here.
- a number of sugar-modified nucleotides are known in the art, such as Tachas et al. US Pat. No. 8,299,039 (especially columns 17-22) or Journal ⁇ ⁇ of Medicinal Chemistry, 2016, 59, pp 9645-9667, Sugar-modified nucleotides described in Medicinal Chemistry Communication, 2014, 5, pp 1454-1471, Future Medicinal Chemistry, 2011, 3, pp 339-365, etc. can also be used as an embodiment of the present invention.
- a person skilled in the art will appropriately select a sugar-modified nucleotide from such sugar-modified nucleotides in consideration of the antisense effect, affinity for the target RNA partial sequence, resistance to nucleolytic enzymes, and the like. Can be used.
- Nucleobase is generally a base component constituting a nucleic acid, and as natural nucleobases, purine bases: adenine (A) and guanine (G), and pyrimidine bases: thymine (T), cytosine (C). And uracil (u).
- a natural nucleobase and its modified nucleobase can be used for the base moiety of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides used in the present specification.
- the modified nucleobase can base pair with any nucleobase (preferably a base complementary to the nucleobase before modification) (ie, form a hydrogen bond).
- modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, adenine and guanine 6-methyl and others Alkyl derivatives, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, pyrimidine base 5-propynyl (—C ⁇ C—CH 3 ) Uracil and cytosine and other alkynyl derivatives, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other Adenine and guanine substituted in position 8 5-halo, especially 5-bromo, 5-trifluoromethyl, 5-
- nucleobases include phenoxazine cytidine (1H-pyrimido [5,4-b] [1,4] benzoxazin-2 (3H) -one), phenothiazine cytidine (1H-pyrimido [5,4-b ], [1,4] benzothiazin-2 (3H) -one), G-clamps such as substituted phenoxazine cytidine (eg 9- (2-aminoethoxy) -H-pyrimido [5,4-b] [ 1,4] benzoxazin-2 (3H) -one), carbazole cytidine (2H-pyrimido [4,5-b] indol-2-one), pyridoindole cytidine (H-pyrido [3 ′, 2 ′: 4 , 5] pyrrolo [2,3-d] pyrimidin-2-one).
- substituted phenoxazine cytidine e
- Modified nucleobases may also include those in which purine or pyrimidine bases are substituted with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. .
- modification of the base moiety in nucleotides which can be used for the base moiety in deoxyribonucleotides, ribonucleotides and sugar modified nucleotides. Amino and hydroxy in the base moiety may be independently protected.
- the base moiety in deoxyribonucleotides, ribonucleotides and sugar modified nucleotides is preferably adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U) and 5-methylcytosine (5- at least one selected from the group consisting of me-C).
- RNase H is generally known as a ribonuclease that recognizes a double strand in which DNA and RNA are hybridized and cleaves the RNA to produce a single-stranded DNA.
- RNase H is not limited to a duplex in which DNA and RNA are hybridized, but can also recognize a duplex in which at least one of a base moiety, a phosphodiester binding moiety and a sugar moiety of DNA and RNA is modified. .
- a duplex in which oligodeoxyribonucleotide and oligoribonucleotide are hybridized can also be recognized.
- DNA can be recognized by RNase H when hybridized with RNA.
- RNA can be cleaved by RNase H when hybridized with DNA.
- at least one of a base moiety, a phosphodiester binding moiety and a sugar moiety is modified in at least one of DNA and RNA.
- RNase H examples include, for example, Nucleic Acids Research, 2014, 42, pp 5378-5389, Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp 2296-2300 (noted above, non-patent Reference 1), Molecular BioSystems, 2009, 5, pp 838-843, Nucleic Acid Therapeutics, 2015, 25, pp 266-274, The Journal of Biological Chemistry, 2004, 279, pp 36317-36326 (non-patent literature mentioned above) 2) etc.
- the RNase H used in the present invention is preferably mammalian RNase H, more preferably human RNase H, and particularly preferably human RNase H1.
- the “gap region” is a region including “at least 4 consecutive nucleotides recognized by RNaseH”, and includes 4 or more consecutive nucleotides, and is not particularly limited as long as it is recognized by RNaseH.
- the nucleotides are preferably independently selected from deoxyribonucleotides and sugar-modified nucleotides.
- the “5 ′ wing region” is a region that includes “at least one nucleotide” that is linked to the 5 ′ side of the gap region and does not include the “at least four consecutive nucleotides recognized by RNaseH”.
- the sugar moiety of the nucleotide at the 3 ′ end of the 5 ′ wing region is different from the sugar moiety of the nucleotide at the 5 ′ end of the gap region.
- the boundary between the 5 'wing region and the gap region is confirmed by the difference in the sugar moiety.
- the 5 ′ terminal nucleotide of the gap region is a deoxyribonucleotide
- the 3 ′ terminal nucleotide of the 5 ′ wing region is a sugar-modified nucleotide.
- the 3 ′ terminal nucleotide of the 5 ′ wing region is Generally, it is a sugar modified nucleotide.
- the 5 ′ wing region is not particularly limited as long as the above definition is satisfied, but the at least one nucleotide is preferably independently selected from deoxyribonucleotide and sugar-modified nucleotide, and includes at least one sugar-modified nucleotide. .
- the “3 ′ wing region” is a region that is linked to the 3 ′ side of the gap region and includes “at least one nucleotide” without including the “at least four consecutive nucleotides recognized by RNaseH”.
- the sugar moiety of the nucleotide at the 5 ′ end of the 3 ′ wing region is different from the sugar moiety of the nucleotide at the 3 ′ end of the gap region. The difference between the sugar moieties confirms the boundary between the 3 'wing region and the gap region.
- the nucleotide at the 3 ′ end of the gap region is a deoxyribonucleotide and the nucleotide at the 5 ′ end of the 3 ′ wing region is a sugar-modified nucleotide.
- the nucleotide at the 5 ′ end of the 3 ′ wing region is: Generally, it is a sugar modified nucleotide.
- the 3 ′ wing region is not particularly limited as long as the above definition is satisfied, but the at least one nucleotide is preferably independently selected from deoxyribonucleotide and sugar-modified nucleotide, and includes at least one sugar-modified nucleotide. .
- An antisense oligonucleotide having a gap region, a 5 'wing region and a 3' wing region is called a gapmer-type antisense oligonucleotide.
- the “central region” is the central region in the oligonucleotide.
- the “5 ′ side region” is a region that is linked to the 5 ′ side of the “central region” and includes at least one nucleotide.
- the “3 ′ side region” is a region that is linked to the 3 ′ side of the “central region” and includes at least one nucleotide.
- the sugar moiety of the nucleotide at the 5 'end of the 3'-end region is different from the sugar moiety of the nucleotide at the 3' end of the central region. Due to the difference in sugar moiety, the boundary between the 3 'heel region and the central region is confirmed.
- the sugar moiety of the 3 'terminal nucleotide of the 5' basal region is different from the sugar moiety of the 5 'terminal nucleotide of the central region. The boundary between the 5 'heel region and the central region is confirmed by the difference in the sugar moiety.
- At least four consecutive nucleotides recognized by RNaseH includes four or more consecutive nucleotides and is not particularly limited as long as it is recognized by RNaseH.
- the number of nucleotides is, for example, 5 to 30, preferably 5 to 15, and more preferably 8 to 12.
- One skilled in the art can determine whether a given at least 4 consecutive nucleotides is “at least 4 consecutive nucleotides recognized by RNase H” by the structure of the sugar moiety of the consecutive nucleotides.
- the antisense oligonucleotide of the present invention does not need to hybridize with the entire target RNA, and may hybridize with at least a part of the target RNA, but usually hybridizes with at least a part of the target RNA.
- an oligonucleotide having an antisense sequence complementary to a partial sequence of the target RNA (DNA, oligodeoxyribonucleotide, or an oligonucleotide usually designed to produce an antisense effect) and at least a part of the target RNA hybridize By doing so, the expression of the target gene is controlled. Further, it is not necessary for the entire antisense oligonucleotide to hybridize, and part of it does not have to hybridize.
- the entire antisense sequence portion may not partially hybridize, but preferably hybridizes.
- the “antisense sequence” means a nucleotide sequence of a nucleotide constituting an oligonucleotide that enables hybridization with a target RNA, and the “antisense sequence portion” refers to the antisense sequence in the oligonucleotide chain. It means a partial structure of a region having an array.
- the complementarity between the antisense sequence portion of the antisense oligonucleotide and the partial sequence of the target RNA is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more (for example, 95%, 96%, 97%, 98%, 99% or more).
- the sequences need not be completely complementary, but more preferably are completely complementary. .
- the antisense oligonucleotide of the present invention has a central region, a 5 ′ region and a 3 ′ region.
- the central region is preferably a gap region
- the 5 ′ side region is preferably a 5 ′ wing region
- the 3 ′ side region is a 3 ′ wing region.
- the central region consists of at least 5 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, and the group consisting of 2'-3'-bridged nucleotides and 3'-modified non-bridged nucleotides At least one sugar-modified nucleotide selected from the group consisting of deoxyribonucleotide, ribonucleotide, 2′-3′-bridged nucleotide, or 3′-position-modified non-bridged nucleotide each independently at its 3 ′ end and 5 ′ end And an oligonucleotide chain composed of at least 4 consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides, 2′-3 ′ bridged nucleotides and 3 ′ modified non-bridged nucleotides, Contains one.
- the number of nucleotides contained in the central region is 5 to 30, preferably 5 to 15, more preferably 8 to 12, and particularly preferably 9 or 10.
- the number of nucleotides contained in the central region is usually selected depending on other factors such as the strength of the antisense effect on the target RNA, low toxicity, cost, and synthesis yield.
- the central region includes at least one sugar-modified nucleotide selected from the group consisting of a 2'-3 'bridged nucleotide and a 3'-position modified non-bridged nucleotide.
- a 2'-3 'bridged nucleotide and a 3'-position modified non-bridged nucleotide are described.
- the partial structure of the 2'-3 'crosslinked nucleotide contained in the central region is preferably represented by the following formula (I).
- Bx is a nucleobase moiety.
- the aforementioned “nucleobase” can be used for the nucleobase moiety.
- X is O or S.
- X is preferably O.
- m is 1, 2, 3 or 4.
- Each of —Q— independently represents —CR 4 R 5 —, —C ( ⁇ O) —, —C ( ⁇ S) —, —C ( ⁇ NR 6 ) —, —O—, —NH—,
- R 1 , R 2 , R 3 , R 4 and R 5 are each independently a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, one or more substitutions C1-C6 alkyl substituted with a group, C2-C6 alkenyl substituted with one or more substituents, C2-C6 alkynyl substituted with one or more substituents, acyl, substituted with one or more substituents Acyl, amide substituted with one or more substituents, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy substituted with one or more substituents, sulfanyl, C1-C6 alkylthio or one or more substitutions A C1-C6 alkylthio substituted with a group; wherein the substituents are each independently a halogen atom, oxo, OJ 1 , NJ 1 J 2 , SJ 1 , azi
- the nucleobase moiety is preferably adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U) and 5-methylcytosine (5-me-C).
- Is at least one selected from the group consisting of R 1 is preferably a hydrogen atom or C1-C3 alkyl, and more preferably a hydrogen atom.
- R 2 , R 3 , R 4 , R 5 , R 7 and R 8 are each independently preferably a hydrogen atom or C1-C3 alkyl, more preferably a hydrogen atom.
- R 6 is preferably C1-C3 alkyl, more preferably methyl.
- m is preferably 1, 2 or 3, more preferably 2 or 3, and particularly preferably 2.
- a preferable partial structure of a 2′-3 ′ bridged nucleotide contained in the central region is represented by the following formula (III).
- Bx is a nucleobase moiety.
- the aforementioned “nucleobase” can be used for the nucleobase moiety.
- X is O or S.
- Bx and R 1 ⁇ R 8 are as defined as X, Bx and R 1 ⁇ R 8 in formula (I), preferable embodiments thereof are also the same.
- -Q 1 -is -O-, -NH-, -NR 6 -or -S-, R 6 is preferably C1-C12 alkyl
- -Q 2- is preferably -CH 2- , More preferably, -Q 1 -is -O- and -Q 2 -is -CH 2- .
- the partial structure of the 3'-modified non-crosslinked nucleotide contained in the central region is preferably represented by the following formula (II).
- Bx is a nucleobase moiety.
- X is O or S.
- R 1 , R 2 , R 3 and R 11 are each independently a hydrogen atom, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkyl substituted with one or more substituents C2-C6 alkenyl substituted with one or more substituents, C2-C6 alkynyl substituted with one or more substituents, acyl, acyl substituted with one or more substituents, one or more substitutions Amido substituted with a group, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy substituted with one or more substituents, sulfanyl, C1-C6 alkylthio or C1-C6 alkylthio substituted with one or more substituents
- R 11 is preferably a hydrogen atom or C1-C3 alkyl, and more preferably a hydrogen atom.
- R 12 is preferably C1-C6 alkyl or C1-C6 alkyl substituted with one or more substituents, more preferably C1-C3 alkyl, and particularly preferably methyl.
- the central region may contain both 2'-3 'bridged nucleotides and 3' modified non-bridged nucleotides.
- the number (total number) of 2′-3 ′ cross-linked nucleotides and 3′-modified non-cross-linked nucleotides contained in the central region is 1 to 30, preferably 1 to 5, more preferably 1 to 2. Particularly preferred is 1.
- the number of 2′-3′-bridged nucleotides and 3′-position-modified non-bridged nucleotides contained in the central region is usually other than the strength of antisense effect against the target RNA, low toxicity, cost, synthetic yield, etc. It is selected according to the element.
- the 2′-3 ′ bridging nucleotide and the 3 ′ modified non-bridging nucleotide contained in the central region can be included anywhere in the central region, but from the third nucleotide counting from the 3 ′ end of the central region. It is preferable to include it between the 5 'end.
- the site containing a 2′-3 ′ bridged nucleotide or a 3′-modified non-bridged nucleotide is usually selected depending on other factors such as the strength of the antisense effect on the target RNA and the low toxicity.
- a sequence portion close to a base that forms a base pair with a mutated base (for example, the fifth counting from the base that forms a base pair with a mutated base)
- the base that forms a base pair with the mutated base is a 2′-3 ′ bridged nucleotide or a 3′-modified non-bridged nucleotide.
- At least one nucleotide is preferably phosphorothioated, more preferably 80% of the nucleotide is phosphorothioated, and 90% of the nucleotide is phosphorothioated are more preferred, and it is particularly preferred that all are phosphorothioated.
- the 5 ′ region consists of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, the 3 ′ end of which is a sugar-modified nucleotide, wherein
- the 3 ′ terminal sugar modified nucleotide is selected from sugar modified nucleotides that bind to the central region, excluding 2′-3 ′ bridged nucleotides and 3 ′ modified non-bridged nucleotides, and deoxyribonucleotides, 2′-3 It does not include an oligonucleotide chain composed of at least 4 consecutive nucleotides independently selected from the group consisting of 'bridged nucleotides and 3'-modified unbridged nucleotides.
- the 3 ′ region consists of at least one nucleotide independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, the 5 ′ end of which is a sugar-modified nucleotide, wherein
- the 5′-terminal sugar-modified nucleotide is selected from sugar-modified nucleotides that bind to the central region, excluding 2′-3′-bridged nucleotides and 3′-modified non-bridged nucleotides, and deoxyribonucleotides, 2′-3 It does not include an oligonucleotide chain composed of at least 4 consecutive nucleotides independently selected from the group consisting of 'bridged nucleotides and 3'-modified unbridged nucleotides.
- the number of nucleotides contained in the 5'-side region is 1 to 15, preferably 1 to 7, more preferably 2 to 5, and particularly preferably 3.
- the number of nucleotides contained in the 5 'region is usually selected depending on other factors such as the strength of antisense effect against the target RNA, low toxicity, cost, and synthesis yield.
- the 3 'side region is the same as the 5' side region.
- the sugar-modified nucleotide contained in the 5 ′ region is preferably a nucleotide having increased affinity for a partial sequence of the target RNA or a nucleotide having increased resistance to a nucleolytic enzyme due to substitution or the like. More preferably, it is independently selected from 2′-modified non-crosslinked nucleotides and 2 ′, 4′-BNA.
- the 2′-modified non-bridging nucleotide is preferably a 2′-O-methylated nucleotide, 2′-O-methoxyethyl (MOE) nucleotide, 2′-O-aminopropyl (AP) nucleotide, 2′- Independently selected from the group consisting of fluorinated nucleotides, 2′-O- (N-methylacetamido) (NMA) and 2′-O-methylcarbamoylethyl (MCE) nucleotides, more preferably 2 ′ It is independently selected from -O-methoxyethyl (MOE) nucleotides and 2'-O-methylcarbamoylethyl (MCE) nucleotides, particularly preferably 2'-O-methoxyethyl (MOE) nucleotides.
- MOE methoxyethyl
- AP aminopropyl
- 2 ′, 4′-BNA is preferably LNA, cEt-BNA, ENA, BNA NC , AmNA and scpBNA, more preferably LNA including a partial structure represented by the following formula (VI).
- the 3 ′ side region is the same as the 5 ′ side region.
- Bx represents a nucleobase moiety and has the same meaning as Bx in formula (I).
- the type, number and position of sugar-modified nucleotides, deoxyribonucleotides and ribonucleotides in the 5'-side region can affect the antisense effect exhibited by the antisense oligonucleotide disclosed herein. Since the type, number, and position differ depending on the target RNA sequence and the like, it cannot be generally stated, but those skilled in the art can determine a preferred embodiment in consideration of the description of the literature relating to the antisense method. . Also, the antisense effect of the oligonucleotide after modification of the base moiety, sugar moiety or phosphodiester binding moiety is measured, and the measured value obtained should not be significantly lower than that of the oligonucleotide before modification.
- the modification is a preferred embodiment.
- the antisense effect is measured by introducing a test oligonucleotide into a cell or the like, and expressing the target RNA controlled by the antisense effect produced by the test oligonucleotide.
- the amount, the expression level of cDNA related to the target RNA, the amount of protein related to the target RNA, etc. can be determined by appropriately using known techniques such as Northern blotting, quantitative PCR, Western blotting, etc. it can.
- the 3 'side region is the same as the 5' side region.
- a preferred form in the 5 ′ region consists of 2 to 5 nucleotides independently selected from the group consisting of 2′-modified non-crosslinked nucleotides, 2 ′, 4′-BNA, and deoxyribonucleotides.
- An oligonucleotide comprising at least two nucleotides selected from the group consisting of modified non-bridging nucleotides and 2 ′, 4′-BNA.
- it is an oligonucleotide composed of 2 to 5 nucleotides independently selected from the group consisting of a 2′-modified non-crosslinked nucleotide and 2 ′, 4′-BNA, and more preferably a 2′-modified An oligonucleotide consisting of 2 to 3 nucleotides independently selected from the group consisting of non-bridging nucleotides and 2 ′, 4′-BNA. More preferably, it is an oligonucleotide consisting of 2 to 3 nucleotides independently selected from LNA and 2′-O-methoxyethyl (MOE) nucleotides, and particularly preferably consisting of 2 to 3 LNAs.
- MOE 2′-O-methoxyethyl
- oligonucleotide it is an oligonucleotide.
- Another preferred form is an oligonucleotide composed of five 2′-modified non-crosslinked nucleotides.
- the 5 ′ region consists of 2 to 5 nucleotides independently selected from the group consisting of 2 ′, 4′-BNA and deoxyribonucleotide, and includes at least two 2 ′, 4 ′.
- -Oligonucleotides containing BNA and such oligonucleotides can be referred to International Publication No. 2016/127002.
- the 3 ′ side region is the same as the 5 ′ side region.
- nucleotides contained in the 5 ′ region are preferably phosphorothioated, more preferably 50% of the nucleotide is phosphorothioated, and 80% of the nucleotide is phosphorothioated. It is even more preferred that all are phosphorothioated. As another form, it is preferable that all of the nucleotides contained in the 5 'region are linked by a phosphodiester bond.
- the 3 'side region is the same as the 5' side region.
- the 3 ′ end of the 5 ′ region and the 5 ′ end of the central region are linked to form a phosphodiester bond or a modified phosphodiester bond.
- the 5 ′ end and the 3 ′ end of the central region are linked to form a phosphodiester bond or a modified phosphodiester bond.
- the 3 ′ end of the 5 ′ region and the 5 ′ end of the central region are linked to form a modified phosphodiester bond
- the 5 ′ end of the 3 ′ region and the 3 ′ end of the central region Are linked to form a modified phosphodiester bond.
- the 3 ′ end of the 5 ′ region and the 5 ′ end of the central region are linked by a phosphorothioate bond, and the 5 ′ end of the 3 ′ side region and the 3 ′ end of the central region are formed by a phosphorothioate bond. Are connected.
- a functional molecule may be bound directly or indirectly to the antisense oligonucleotide of the present invention.
- the binding between the functional molecule and the antisense oligonucleotide may be direct or indirect via another substance, but the oligonucleotide and the functional molecule are bound by a covalent bond, an ionic bond or a hydrogen bond. It is preferable. From the viewpoint of high stability of the bond, it is more preferable that the bond is directly bonded by a covalent bond, or the bond is bonded by a covalent bond via a linker (linking group).
- the functional molecule When the functional molecule binds covalently to the antisense oligonucleotide, the functional molecule is directly or indirectly linked to the 3 ′ end or 5 ′ end of the antisense oligonucleotide molecule. It is preferable.
- the bond between the linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide molecule is selected according to the functional molecule.
- the linker or functional molecule and the terminal nucleotide of the antisense oligonucleotide molecule are preferably linked by a phosphodiester bond or a modified phosphodiester bond, and more preferably linked by a phosphodiester bond. .
- the linker or functional molecule may be directly linked to the 3′-position oxygen atom of the 3′-end nucleotide of the antisense oligonucleotide molecule or the 5′-position oxygen atom of the 5′-end nucleotide.
- the structure of the “functional molecule” is not particularly limited, and a desired function is imparted to the antisense oligonucleotide by binding. Desired functions include a labeling function, a purification function, and a delivery function to a target site. Examples of molecules that impart a labeling function include compounds such as fluorescent proteins and luciferases. Examples of molecules imparting a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.
- a molecule having a function of delivering an antisense oligonucleotide to a target site is bound as a molecule.
- the molecule having the delivery function is, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp 78-92, Expert Opinion on Drug Delivery, 2014, 11, You can refer to pp822791-822.
- Examples of molecules imparting a function of delivering to a target RNA include lipids and sugars from the viewpoint that antisense oligonucleotides can be efficiently delivered to the liver and the like with high specificity.
- Such lipids include cholesterol; fatty acids; vitamin E (tocopherols, tocotrienols), fat-soluble vitamins such as vitamin A, vitamin D, and vitamin K; intermediate metabolites such as acylcarnitine and acyl CoA; glycolipids; glycerides Their derivatives and the like.
- cholesterol and vitamin E (tocopherols, tocotrienols) are preferable from the viewpoint of higher safety.
- tocopherols are more preferable, tocopherol is further preferable, and ⁇ -tocopherol is particularly preferable.
- the sugar include sugar derivatives that interact with the asialoglycoprotein receptor.
- “Asialoglycoprotein receptor” is present on the surface of liver cells and has the action of recognizing galactose residues of asialoglycoprotein and incorporating the molecule into the cell for degradation.
- the “sugar derivative that interacts with asialoglycoprotein receptor” is preferably a compound having a structure similar to that of a galactose residue and taken into cells by interaction with asialoglycoprotein receptor.
- GalNAc N— Acetylgalactosamine
- examples of the “functional molecule” include sugars (eg, glucose, sucrose, etc.).
- examples of antisense oligonucleotides can be delivered to the organs with high specificity and efficiency by interacting with various proteins on the cell surface of each organ, as a ⁇ functional molecule '', a receptor ligand, Antibodies, peptides or proteins of their fragments.
- the linker is not particularly limited as long as it is a linker.
- Examples of the linker include a group derived from an oligonucleotide having 1 to 20 nucleotides, a group derived from a polypeptide having 2 to 20 amino acids, alkylene having 2 to 20 carbon atoms, and 2 to 20 carbon atoms. And alkenylene.
- the group derived from an oligonucleotide having 2 to 20 nucleotides is a group obtained by removing hydroxy, hydrogen atom, etc. from an oligonucleotide having 2 to 20 nucleotides.
- the group derived from the oligonucleotide having 1 to 20 nucleotides for example, International Publication No. 2017/053995 can be referred to.
- WO 2017/053995 describes, for example, a 3-base linker having a TCA motif, a 1-5 base linker not having a TCA motif, and the like.
- the group derived from a polypeptide having 2 to 20 amino acids is a group obtained by removing hydroxy, hydrogen atom, amino and the like from a polypeptide having 2 to 20 amino acids.
- the linker is preferably C2-C20 alkylene or C2-C20 alkenylene (the methylenes contained in the alkylene and alkenylene are each independently unsubstituted, halogen, hydroxy, protected hydroxy, oxo and thioxo) Substituted with 1 or 2 substituents selected from the group consisting of, and the alkylene and alkenylene methylenes are each independently not substituted or —O—, —NR B — ( R B is a hydrogen atom, C1-C6 alkyl or halo C1-C6 alkyl), substituted by —S—, —S ( ⁇ O) — or —S ( ⁇ O) 2 —).
- the linker is —C ( ⁇ O) —O—, —O—C ( ⁇ O) —NR 13 —
- R 13 is a hydrogen atom, C1-C6 alkyl or halo.
- C1-C6 alkyl —C ( ⁇ O) —NR 13 —
- R 13 represents a hydrogen atom, C1-C6 alkyl or halo C1-C6 alkyl
- R 13 represents a hydrogen atom, C1-C6 alkyl or halo C1-C6 alkyl), —NR 13 —C ( ⁇ O) —NR 13 — (R 13 is independently a hydrogen atom, C1- A group represented by C6 alkyl or halo C1-C6 alkyl) and the like.
- the linker is more preferably C2-C20 alkylene (the alkylene methylenes are each independently not substituted or replaced by -O-. Each unsubstituted methylene is independently , Unsubstituted or substituted with hydroxy or protected hydroxy), more preferably C8-C12 alkylene, wherein the methylenes of the alkylene are each independently unsubstituted or Each of the unsubstituted methylenes is independently unsubstituted or substituted with hydroxy), particularly preferably 1,8-octylene.
- the linker is particularly preferably a group represented by the following formula (VII).
- one * represents a bonding position with a group derived from an oligonucleotide (atom constituting a nucleotide), and the other * represents a bonding position with a group derived from a functional molecule (derived from a functional molecule).
- the linker is more preferably a C2-C20 alkylene (wherein the methylenes of the alkylene are each independently not substituted or —O— or —NR B — (R B is a hydrogen atom) Or each of the unsubstituted methylenes is independently unsubstituted or substituted by oxo, and more preferably the following formula: (Wherein e is each independently an integer of 1 to 6), particularly preferably the following formula: It is group represented by these.
- the protecting group of the “protected hydroxy” is not particularly limited as long as it is stable when binding the functional molecule and the oligonucleotide.
- the linker is not particularly limited. For example, any of those described in Protective Groups Organic Synthesis 4th edition, T. W. Greene, P. G. M. Wuts, John Wiley & Sons Inc. (2006) Can be mentioned.
- C1-C6 alkyl for example, methyl, t-butyl and the like
- triarylmethyl for example, triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl and the like
- Ether-based protecting groups such as methoxymethyl, methylthiomethyl, methoxyethyl, benzyloxymethyl, 2-tetrahydropyranyl, ethoxyethyl, etc .
- acyl for example, formyl, acetyl, pivaloyl, benzoyl
- An acyl protecting group such as tri (C1-C6 alkyl) silyl (for example, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, dimethylisopropylsilyl, etc.), (C1- C6 al ) Diaryls
- the protecting group for “protected hydroxy” is preferably benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triphenylmethyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthene -9-yl or 9- (p-methoxyphenyl) xanthen-9-yl, more preferably monomethoxytrityl, dimethoxytrityl or trimethoxytrityl, and even more preferably dimethoxytrityl.
- the present invention also includes prodrugs of the antisense oligonucleotides.
- a prodrug is a derivative of a pharmaceutical compound having a group that can be chemically or metabolically decomposed, and is a pharmacologically active pharmaceutical compound by solvolysis or degradation in vivo under physiological conditions. It is a compound derived from Methods for selecting and producing suitable prodrug derivatives are described, for example, in Design of Prodrugs (Elsevier, Amsterdam, 1985).
- a prodrug such as an acyloxy derivative produced by reacting the compound with a suitable acyl halide, a suitable acid anhydride or a suitable halogenated alkyloxycarbonyl compound.
- Particularly preferred structures as prodrugs include —O—C ( ⁇ O) C 2 H 5 , —O—C ( ⁇ O) (t—Bu), —O—C ( ⁇ O) C 15 H 31 , — O—C ( ⁇ O) ⁇ (m—CO 2 Na—Ph), —O—C ( ⁇ O) CH 2 CH 2 CO 2 Na—OC ( ⁇ O) CH (NH 2 ) CH 3 , —O— And C ( ⁇ O) CH 2 N (CH 3 ) 2 or —O—CH 2 OC ( ⁇ O) CH 3 .
- the compound having the amino group is reacted with an appropriate acid halide, an appropriate mixed acid anhydride, or an appropriate halogenated alkyloxycarbonyl compound.
- Prodrugs to be produced are exemplified.
- oligonucleotide comprising a ribonucleotide (eg, oligoribonucleotide nucleotide, RNA), an oligonucleotide comprising a peptide nucleic acid (PNA), complementary to an antisense oligonucleotide, Or a double-stranded oligonucleotide comprising an oligonucleotide comprising deoxyribonucleotides (eg, oligodeoxyribonucleotide, DNA) (eg, WO2013 / 088983, WO2017 / 068791, WO2017 / 068790 or WO2018 / 003739), a single-stranded oligonucleotide in which an RNA oligonucleotide complementary to an antisense oligonucleotide is linked by a linker (for example, international Hirakidai is described
- the linker is not limited to those described in International Publication No. 2017/131124, and may include, for example, a non-nucleotide structure. Moreover, the single-stranded oligonucleotide which the RNA oligonucleotide complementary with the antisense oligonucleotide was connected directly is also mentioned.
- An oligonucleotide complex comprising (i) the antisense oligonucleotide and (ii) an oligonucleotide comprising at least one ribonucleotide and comprising a region that hybridizes to the antisense oligonucleotide (i).
- (B) (I) a group derived from the antisense oligonucleotide; and (ii) a group derived from an oligonucleotide containing at least one ribonucleotide and including a region that hybridizes to the antisense oligonucleotide (i). And (i) a group derived from an antisense oligonucleotide and a group derived from the oligonucleotide (ii) linked to each other.
- a group derived from an antisense oligonucleotide and (ii) a group derived from an oligonucleotide may be linked by a group derived from an oligonucleotide that is degraded under physiological conditions. , May be linked by a linking group containing a non-nucleotide structure, or may be linked directly.
- (C) (Iii) an oligonucleotide in which an oligonucleotide chain containing at least one ribonucleotide is linked to a group derived from the antisense oligonucleotide, and (iv) at least 4 consecutive nucleotides recognized by RNaseH
- An oligonucleotide complex comprising an oligonucleotide comprising an oligonucleotide chain comprising: An oligonucleotide wherein said oligonucleotide chain comprising at least one ribonucleotide of (iii) and said oligonucleotide chain comprising at least 4 consecutive nucleotides recognized by RNase H of (iv) hybridize; Complex.
- a group derived from an oligonucleotide comprising an oligonucleotide chain comprising a nucleotide, wherein (iii) the group derived from the oligonucleotide and (iv) a group derived from the oligonucleotide are linked,
- An oligonucleotide wherein the oligonucleotide chain comprising at least one ribonucleotide of (iii) and an oligonucleotide chain comprising at least 4 consecutive nucleotides recognized by RNaseH of (iv) hybridize.
- a group derived from an oligonucleotide and (iv) a group derived from an oligonucleotide may be linked by a group derived from an oligonucleotide that is degraded under physiological conditions. They may be linked by a linking group containing a nucleotide structure, or may be linked directly.
- a group derived from an antisense oligonucleotide and an oligonucleotide chain containing at least one ribonucleotide are linked by a group derived from an oligonucleotide that is degraded under physiological conditions. It may be linked by a linking group containing a non-nucleotide structure, or may be linked directly.
- oligonucleotide that is degraded under physiological conditions may be any oligonucleotide that can be degraded by enzymes such as various DNases (deoxyribonucleases) and RNases (ribonucleases) under physiological conditions. In some or all of them, a base, sugar, or phosphate bond may or may not be chemically modified.
- the “oligonucleotide that is degraded under physiological conditions” is, for example, an oligonucleotide containing at least one phosphodiester bond, preferably linked by a phosphodiester bond, more preferably an oligodeoxyribonucleotide or oligoribonucleotide.
- Oligonucleotides that are degraded under physiological conditions may or may not include partially complementary sequences within oligonucleotides that are degraded under physiological conditions, but preferably are partially Does not contain a complementary sequence.
- Examples of such oligonucleotides are (N) k ′ linked by phosphodiester bonds, where N is independently adenosine, uridine, cytidine, guanosine, 2′-deoxyadenosine, thymidine, 2′-deoxycytidine. Or 2′-deoxyguanosine, and k is an integer of 1 to 40 (the number of repetitions).
- k ′ is preferably 3 to 20, more preferably 4 to 10, still more preferably 4 to 7, even more preferably 4 or 5, and particularly preferably 4.
- the “linking group containing a non-nucleotide structure” is a linking group having at least one “non-nucleotide structure” as a structural unit.
- Examples of the non-nucleotide structure include a structure having no nucleobase.
- the “linking group containing a non-nucleotide structure” may or may not contain a nucleotide (deoxyribonucleoside group, ribonucleoside group, etc.).
- the “linking group including a non-nucleotide structure” is, for example, a group having the following structure.
- V 11 is C2-C50 alkylene (the C2-C50 alkylene, or an unsubstituted, substituted with one or more substituents independently selected from substituent group V a),
- Rb is a halogen atom, hydroxy, amino, C1-C6 alkoxy, C1-C6 alkoxy substituted with C1-C6 alkoxy or carbamoyl, mono-C alkylamino, di-C1-C6 alkylamino or C-alkyl group
- Rc is A hydrogen atom, C1-C6 alkyl, halo C1-C6 alkyl, C1-C6 alkylcarbonyl, halo C1-C6 alkylcarbonyl, C1-C6 alkoxycarbonyl, C1-C6 alkoxycarbonyl or C1-C6 alkoxycarbonyl substituted with carbamoyl, Mono C1-C6 alkylaminocarbonyl, di-C1-C6 alkylamin
- ком ⁇ онент 1 is preferably an integer of 1 to 30, and p 1 is preferably an integer of 1 to 30.
- q 11 is preferably an integer of 1 to 6, more preferably an integer of 1 to 3.
- q 12 is preferably an integer of 1 to 6, more preferably an integer of 1 to 3.
- P 11 is preferably —P ( ⁇ O) (OH) —.
- the type, number, and position of sugar-modified nucleotides, deoxyribonucleotides, and ribonucleotides can affect the antisense effect and the like exhibited by the prodrugs of the antisense oligonucleotides disclosed herein. Since the type, number, and position differ depending on the target RNA sequence and the like, it cannot be generally stated, but those skilled in the art can determine a preferred embodiment in consideration of the description of the literature relating to the antisense method. .
- the antisense effect of the antisense oligonucleotide prodrug after modification of the base moiety, sugar moiety or phosphodiester binding moiety is measured, and the measured value obtained is the value of the antisense oligonucleotide prodrug before modification. If it is not significantly reduced (for example, if the measurement value of the prodrug of the antisense oligonucleotide after modification is 30% or more of the measurement value of the prodrug before modification), the modification is a preferred embodiment Can be evaluated.
- the antisense effect is measured by introducing a test oligonucleotide into a cell or the like, and expressing the target RNA controlled by the antisense effect produced by the test oligonucleotide.
- the amount, the expression level of cDNA related to the target RNA, the amount of protein related to the target RNA, etc. can be determined by appropriately using known techniques such as Northern blotting, quantitative PCR, Western blotting, etc. it can.
- the group derived from the oligonucleotide (ii) in the oligonucleotide complex shown in (A) or the oligonucleotide (ii) in the oligonucleotide shown in (B) is a ribonucleotide, deoxyribonucleotide, sugar moiety modification It is independently selected from nucleotides, preferably selected from ribonucleotides.
- the ribonucleotides are preferably linked to each other by phosphodiester bonds.
- the oligonucleotide of (ii) or a group derived from the oligonucleotide is selected from ribonucleotides and sugar-modified nucleotides, and the sugar-modified nucleotides are 2′-3′-bridged nucleotides and 3′-position-modified nucleotides. It is selected from sugar-modified nucleotides excluding non-crosslinking nucleotides. At this time, it is preferable that the terminal of the oligonucleotide is at least one sugar moiety-modified nucleotide.
- This sugar-modified nucleotide is preferably a 2′-O-methylated nucleotide, and preferably linked to an adjacent nucleotide by a phosphorothioate bond.
- an oligonucleotide chain comprising at least one ribonucleotide among the oligonucleotides in (iii) (at least 4 recognized by RNaseH) This also applies to the portion that hybridizes with the oligonucleotide chain containing the consecutive nucleotides.
- the nucleotides at the 3 'end and 5' end of the oligonucleotide of (ii) are preferably sugar-modified nucleotides.
- the nucleotide at the end not bound to the group derived from the antisense oligonucleotide is preferably , A sugar-modified nucleotide.
- the nucleotide at the end not bound to the group derived from the antisense oligonucleotide is Preferably, it is a sugar moiety-modified nucleotide.
- the number of bases in the oligonucleotide (ii) or the group derived from the oligonucleotide is not particularly limited, and may be the same as or different from the number of bases in the (i) antisense oligonucleotide (or group derived from). .
- the number of bases of the oligonucleotides (i) and (ii) is preferably the same, and the whole oligonucleotides (i) and (ii) preferably hybridize.
- the group derived from the oligonucleotide (iv) in the oligonucleotide complex shown in (C) and the oligonucleotide (iv) in the oligonucleotide in (D) are ribonucleotides, deoxyribonucleotides, sugar-modified nucleotides And is preferably selected from deoxyribonucleotides and sugar-modified nucleotides.
- the sugar moiety-modified nucleotide contained in the oligonucleotide (iv) or the group derived from the oligonucleotide is preferably selected from sugar moiety-modified nucleotides excluding 2′-3 ′ bridged nucleotides and 3′-position-modified non-bridged nucleotides .
- the at least one sugar moiety-modified nucleotide is preferably at least one selected from 2′-modified non-crosslinked nucleotides and 2 ′, 4′-BNA, more preferably 2′-O-methylated nucleotides.
- the nucleotides contained in (iv) or the oligonucleotide of the group derived from the oligonucleotide are preferably linked to each other by a phosphorothioate bond.
- the oligonucleotide complex shown in (A) has a functional molecule
- the oligonucleotide of (ii) includes a functional molecule
- the functional molecule is a terminal of the oligonucleotide of (ii) It is preferable to bind to.
- the oligonucleotide shown in (B) has a functional molecule
- the oligonucleotide complex shown in (iv) contains a functional molecule, and the functional molecule is a terminal of the oligonucleotide of (iv). It is preferable to bind to.
- the preferred embodiment of the functional molecule and its binding is as described above.
- the oligonucleotide or the group derived from the oligonucleotide (ii) may further have a group derived from the antisense oligonucleotide.
- the group derived from the antisense oligonucleotide of (ii) may be the same as or different from the group derived from the antisense oligonucleotide or oligonucleotide of (i). Further, it may or may not be a group derived from the antisense oligonucleotide of the present invention.
- the group derived from the antisense oligonucleotide (ii) preferably does not hybridize with the antisense oligonucleotide or group derived from the antisense oligonucleotide (i).
- the group derived from the oligonucleotide or oligonucleotide of (iv) may be a group derived from an antisense oligonucleotide or an antisense oligonucleotide.
- the antisense oligonucleotide or the group derived from the antisense oligonucleotide of (iv) may be the same as or different from the group derived from the antisense oligonucleotide contained in the oligonucleotide of (iii). Further, it may or may not be a group derived from the antisense oligonucleotide of the present invention. It is preferable that the antisense oligonucleotide or the group derived from the antisense oligonucleotide (iv) does not hybridize with the antisense oligonucleotide or the group derived from the antisense oligonucleotide (iii). Examples of antisense oligonucleotides that are not antisense oligonucleotides of the present invention include the following antisense oligonucleotides.
- an antisense oligonucleotide having a central region, a 5 ′ region and a 3 ′ region, wherein -The central area is Consisting of at least 5 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides, wherein the sugar-modified nucleotides are 2′-3′-bridged nucleotides and 3′-position-modified non-bridged nucleotides Selected from sugar-modified nucleotides excluding An oligonucleotide chain composed of at least 4 consecutive nucleotides, the 3 ′ end and 5 ′ end of which are independently deoxyribonucleotides or ribonucleotides and independently selected from deoxyribonucleotides; Including at least one; -5 'side area is Consisting of at least one nucleotide independently selected from the group consisting of deoxyribonucleot
- the central region is preferably a gap region
- the 5 ′ side region is preferably a 5 ′ wing region
- the 3 ′ side region is preferably 3 ′. Wing area.
- preferred embodiments of the 5 'side region and the 3' side region are the same as the 5 'side region and the 3' side region in the antisense oligonucleotide of the present invention.
- a preferred embodiment of the central region is that the central region in the antisense oligonucleotide of the present invention does not contain a sugar moiety-modified nucleotide selected from the group consisting of a 2′-3 ′ bridged nucleotide and a 3′-modified non-bridged nucleotide. It is the same.
- antisense oligonucleotides consisting of at least 5 nucleotides independently selected from the group consisting of deoxyribonucleotides, ribonucleotides and sugar-modified nucleotides; Does not include an oligonucleotide chain composed of at least 4 consecutive nucleotides independently selected from the group consisting of deoxyribonucleotides, 2′-3 ′ bridged nucleotides, and 3′-modified unbridged nucleotides, Antisense oligonucleotide.
- a linking group containing a non-nucleotide structure and an oligonucleotide can be bound by a general amidite method or H-phosphonate method.
- an amiditization reagent eg, 2-cyanoethyl chloro (diisopropylamino) phosphinite, 2-cyanoethyl bis (diisopropylamino) phosphinite
- an H-phosphonate reagent for example, diphenyl phosphite, phosphorous acid, etc.
- the nucleotides can be further extended using a commercially available automated nucleic acid synthesizer.
- the compound having the two hydroxy groups is a protective / deprotection reaction known to those skilled in the art (for example, see Protective / Groups / Organic / Synthesis / 4th edition), oxidation reaction, reduction reaction, condensation reaction (oxidation reaction, reduction reaction). Reactions and condensation reactions are used in combination with, for example, Comprehensive Organic Transformations, 2nd edition, RCLarock, Wiley-VCH (1999), etc., for example, raw materials such as amino acids, carboxylic acids, diol compounds, etc. Can be synthesized.
- the linking group containing a non-nucleotide structure has a functional group other than the two hydroxy groups (for example, an amino group, a hydroxy group, or a thiol group), a protecting group known to those skilled in the art (for example, Protective Groups Organic Synthesis). It can be efficiently extended by protecting with 4th edition).
- a protecting group known to those skilled in the art for example, Protective Groups Organic Synthesis. It can be efficiently extended by protecting with 4th edition).
- WO2012 / 017919, WO2013 / 103146, WO2013 / 133221, WO2015 / 099187, WO2016 / 104775 etc. can be referred for the synthesis
- a linking group containing a non-nucleotide structure can be bound.
- An example of the synthesis method is shown below.
- a partial structure having a functional group such as an amino group is bonded to the 5 ′ end of the oligonucleotide by a method known to those skilled in the art (for example, 6- (trifluoroacetylamino) hexyl-[(2-cyanoethyl)-(N , N-diisopropyl)]-phosphoramidite, etc.)
- a partial structure having a functional group such as an amino group is attached to the 3 ′ end of another oligonucleotide by a method known to those skilled in the art (for example, 2 -((4,4'-dimethoxytrityl) oxymethyl) -6-fluorenylmethoxycarbonylamino-hexane-1-succinoyl-long chain alkyla
- Two oligonucleotides can be linked by converting the two functional groups of a linking group containing a non-nucleotide structure into a desired functional group that reacts with the amino group or the like.
- two functional groups possessed by a linking group containing a non-nucleotide structure are carboxylic acid, ester, active ester (N-hydroxysuccinimidation, etc.), acid chloride, activated carbonic acid diester (4-nitrophenylated carbonic acid diester, etc.) After conversion to isocyanate, etc., the reaction can be carried out by reacting under known N-carbonylation conditions.
- Antisense oligonucleotides or prodrugs thereof include those existing as a mixture or mixture of isomers in addition to their tautomerism and geometric isomerism.
- an asymmetric center when an asymmetric center is present or when an asymmetric center is generated as a result of isomerization, it includes those present as a mixture of the respective optical isomers and arbitrary ratios.
- diastereomers by respective optical isomerism also exist.
- the present invention also includes those containing all these types in any proportion.
- the optically active substance can be obtained by a method well known for this purpose.
- the antisense oligonucleotide of the present invention or a prodrug thereof contains a modified phosphodiester bond (for example, phosphorothioate bond) and the phosphorus atom is an asymmetric atom
- the oligo of the phosphorus atom having a controlled stereo structure
- Any form of oligonucleotide in which the steric configuration of the nucleotide and phosphorus atoms is not included is within the scope of the present invention.
- the antisense oligonucleotides, prodrugs or pharmaceutically acceptable salts thereof of the present invention can exist in any crystalline form depending on the production conditions, and can exist as any hydrate, These crystal forms and hydrates and mixtures thereof are also included in the scope of the present invention. Further, it may exist as a solvate containing an organic solvent such as acetone, ethanol, 1-propanol or 2-propanol, but any of these forms is included in the scope of the present invention.
- the antisense oligonucleotide of the present invention or a prodrug thereof can be converted into a pharmaceutically acceptable salt or released from the produced salt, if necessary.
- pharmaceutically acceptable salts of antisense oligonucleotides or prodrugs thereof include alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, organic bases (triethylamine, Trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid) , Methanesulfonic acid, p-toluenesulfonic acid and the like).
- a partial structure represented by —P ( ⁇ O) (OH) — is converted into an anionic partial structure represented by —P ( ⁇ O) (O ⁇ ) —, and an alkali metal (lithium, Sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, etc. may form salts.
- a partial structure represented by —P ( ⁇ O) (SH) — that forms a phosphorothioate bond is converted into an anionic partial structure represented by —P ( ⁇ O) (S ⁇ ) —.
- a salt may be formed with an alkali metal, an alkaline earth metal, ammonium or the like. The same applies to other modified phosphodiester bonds.
- the antisense oligonucleotide of the present invention or a prodrug thereof can be prepared by a person skilled in the art by appropriately selecting a known method.
- a person skilled in the art designs a nucleotide sequence of an antisense oligonucleotide based on information on the nucleotide sequence of a target RNA, and commercially available automatic nucleic acid synthesizers (Applied Biosystems, Beckman, Gene Design, etc.) Can be synthesized. It can also be synthesized by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzyme. That is, the method for producing an antisense oligonucleotide or a prodrug thereof according to the present embodiment can include a step of extending a nucleotide chain at the 3 'end or the 5' end.
- An antisense oligonucleotide or a prodrug thereof can be prepared by purifying the resulting oligonucleotide by reverse phase column chromatography or the like.
- the antisense oligonucleotide or prodrug thereof of the present invention can effectively control the expression of the target gene. Therefore, the present invention can provide a composition containing the antisense oligonucleotide of the present invention as an active ingredient, for example, for controlling the expression of a target gene by the antisense effect.
- the antisense oligonucleotide of the present invention or a prodrug thereof can obtain a high drug effect by administration at a low concentration, and can treat, prevent, or prevent diseases associated with increased expression of target genes such as metabolic diseases, tumors, and infectious diseases.
- a pharmaceutical composition for improvement may also be provided in some embodiments.
- composition containing the antisense oligonucleotide of the present invention or a prodrug thereof can be formulated by a known pharmaceutical method.
- a known pharmaceutical method for example, capsule, tablet, pill, liquid, powder, granule, fine granule, film coating, pellet, troche, sublingual, chewing agent, buccal, paste, syrup, suspension, As elixirs, emulsions, coatings, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories, etc. enteral (oral) or non-enteric Can be used enterally.
- pharmacologically or food and drink acceptable carriers specifically, sterilized water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspensions, surfactants, pH regulators. , Stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, tonicity agent, soothing agent, bulking agent, disintegrant, buffering agent, coating agent, lubricant, It can be appropriately combined with a colorant, sweetener, thickener, flavoring agent, solubilizing agent or other additives.
- the administration form of the composition containing the antisense oligonucleotide of the present invention or a prodrug thereof is not particularly limited, and examples thereof include enteral (oral) and non-enteral administration. More preferably, intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, nasal administration and intravitreal administration Etc. and administration by infusion.
- Diseases that can be treated, prevented, or ameliorated by the nucleic acid drug using the antisense oligonucleotide of the present invention or a prodrug thereof are not particularly limited.
- metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammation examples include diseases caused by gene expression, such as sex diseases, autoimmune diseases, and inherited rare diseases.
- hypercholesterolemia hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama congenital muscular dystrophy, Woolrich congenital muscular dystrophy, merosin) Deficient congenital muscular dystrophy, integrin deficiency, Walker-Warburg syndrome, etc.), Becker muscular dystrophy, limb-girdle muscular dystrophy, miyoshi type muscular dystrophy, facial scapulohumeral muscular dystrophy, etc.), Huntington's disease, Alzheimer's disease, transthyretin amyloidosis , Familial amyloid cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type 2 diabetes, chronic nephropathy, RS virus infection, Ebola hemorrhagic fever, Marble Fever, HIV, influenza, hepatitis B, hepatitis C
- various other mammalian diseases can be treated, prevented and ameliorated by the composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof.
- the composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof.
- a composition comprising an antisense oligonucleotide can also be applied to other species such as birds (eg, chickens).
- the dose or intake is determined by the subject's age, weight, symptom, health condition, The dose or intake is appropriately selected according to the type (pharmaceutical, food and drink, etc.), and the dose or intake is 0.0001 mg / kg / day to 100 mg / kg / day in terms of antisense oligonucleotide. preferable.
- the antisense oligonucleotide of the present invention or a prodrug thereof can reduce toxicity as compared with the conventional antisense oligonucleotide while effectively controlling the expression of the target gene. Therefore, the antisense oligonucleotide of the present invention or a prodrug thereof can be administered to animals including humans, and a method for more safely controlling the expression of the target gene by the antisense effect can be provided.
- a method for treating, preventing or improving various diseases associated with increased expression of a target gene which comprises administering a composition containing the antisense oligonucleotide of the present invention or a prodrug thereof to an animal including a human. Can also be provided.
- Preferred methods using the antisense oligonucleotide of the present invention include those shown below.
- a method for controlling the function of a target RNA comprising a step of bringing a cell into contact with the antisense oligonucleotide of the present invention or a prodrug thereof.
- a method for controlling the function of a target RNA in a mammal comprising a step of administering a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof to the mammal.
- a method for controlling the expression of a target gene comprising a step of contacting a cell with an antisense oligonucleotide of the present invention or a prodrug thereof.
- a method for controlling the expression of a target gene in a mammal comprising a step of administering a pharmaceutical composition comprising the antisense oligonucleotide of the present invention or a prodrug thereof to the mammal.
- Use of the antisense oligonucleotide of the present invention or a prodrug thereof for controlling the expression of a target gene in a mammal Use of the antisense oligonucleotide of the present invention or a prodrug thereof for the manufacture of a medicament for controlling the expression of a target gene in a mammal.
- Control of the function of the target RNA in the present invention is achieved by regulating or converting a splicing function such as translational inhibition or exon skipping, which occurs when an antisense sequence part coats a part of the target RNA by hybridization, or It means that the function of the target RNA is suppressed by the degradation of the target RNA, which can be caused by recognizing the part where the antisense sequence part and a part of the target RNA are hybridized.
- a splicing function such as translational inhibition or exon skipping
- the mammal is preferably a human.
- the route of administration is preferably enteral. In other embodiments, the route of administration is parenteral.
- the 2′-3′-bridged nucleotide and the 3′-position-modified non-bridged nucleotide according to the embodiment of the present invention can be produced by the methods shown in the following order, but the following production method shows an example of a general production method.
- the production method of the 2′-3 ′ bridged nucleotide and the 3′-position modified non-bridged nucleotide according to this embodiment is not limited.
- the raw material compounds when their specific production method is not described, those commercially available can be used, or can be produced according to a known method or a method analogous thereto.
- each of P 1 and P 2 independently represents a hydroxy protecting group
- L G1 represents a leaving group
- —Q— represents —CR 4 R 5 —, —C ( ⁇ O) —, — C ( ⁇ S) — or —C ( ⁇ NR 6 ) —
- R 4 , R 5 , R 6 , and other symbols are the same as defined above.
- the leaving group includes acetate (AcO), p-nitrobenzoate (PNBO), sulfonate (for example, methanesulfonate (mesylate: MsO), p-toluenesulfonate (tosylate: TsO), p-bromobenzenesulfonate (brosylate). : BsO), p-nitrobenzenesulfonate (nosylate: NsO), fluoromethanesulfonate, difluoromethanesulfonate, trifluoromethanesulfonate (triflate: TfO) and ethanesulfonate) and halogen atoms.
- AcO acetate
- PNBO p-nitrobenzoate
- sulfonate for example, methanesulfonate (mesylate: MsO), p-toluenesulfonate (tosylate: TsO),
- Compound A which is a starting material, can be synthesized, for example, by converting 2 'hydroxy of a ribonucleoside in which 3' and 5 'hydroxy are protected to a leaving group. Conversion to a leaving group can be performed, for example, by sulfonated alcohol (for example, methanesulfonated, p-toluenesulfonated). Chloromethanesulfonic acid or p-toluenesulfonic acid is converted to an appropriate base (for example, , Triethylamine or N, N-dimethyl-4-aminopyridine).
- sulfonated alcohol for example, methanesulfonated, p-toluenesulfonated
- Chloromethanesulfonic acid or p-toluenesulfonic acid is converted to an appropriate base (for example, , Triethylamine or N, N-dimethyl-4-aminopyridine).
- Compound A having various R 1 and R 2 is, for example, described in the following protection / deprotection reaction known to those skilled in the art from compound A-1 described below (for example, described in the Protective Groups in Organic Synthesis 4th edition). Reaction), oxidation reaction, reduction reaction (for example, see Comprehensive Organic Transformations, 2nd edition, RCLarock, Wiley-VCH (1999) etc.) can be used in combination.
- P 3 represents a hydroxy protecting group, and other symbols are the same as defined above.
- compound A in which at least one of R 1 and R 2 is an alkyl group first, hydroxy at the 3 ′ position is protected by hydroxy protection / deprotection reaction, and hydroxy at the 5 ′ position is deprotected. To give the compound (compound A-2). Next, by oxidizing the hydroxy at the 5 'position of Compound A-2, it can introduce the desired R 1 with an alkyl metal reagent or Grignard reagent corresponding to R 1. If also desired, oxidizing the hydroxy again 5 'position, it can introduce the desired R 2 with an alkyl metal reagent, a metal hydride or Grignard reagent corresponding to R 2. By deprotecting the protected hydroxy at the 3 ′ position of the obtained compound, compound A in which at least one of R 1 and R 2 is an alkyl group can be synthesized.
- Cyclization -Q- is -CR 4 R 5 - when it is, can be synthesized cyclized (C) by cyclopropanation reactions generally known.
- C cyclized
- —Q— is —C ( ⁇ O) —, for example, by reacting protected hydroxydiiodomethane with diethylzinc, then deprotecting the protected hydroxy and oxidizing, (C) can be synthesized.
- P 1 and P 2 are hydroxy protecting groups
- L G1 and L G2 are each independently a leaving group
- Q 11 is O
- NH or NR 6 and H is a hydrogen atom.
- K is an integer of 0 to 3
- R 6 and other symbols are the same as defined above.
- Compound D as a starting material is prepared according to methods known to those skilled in the art, such as the method described in Journal of the American Chemical Society, 1998, 120, p 5458, Journal of the Chemical Society, Perkin Transaction 1, 1999, p 2543. Can be synthesized.
- the compound D having various R 1 and R 2 is, for example, described in the following protection / deprotection reaction known to those skilled in the art from the compound D-1 described below (for example, described in the Protective Groups in Organic Synthesis 4th edition). Reaction), oxidation reaction, reduction reaction (for example, see Comprehensive Organic Transformations, 2nd edition, RCLarock, Wiley-VCH (1999)) can be used in combination.
- the specific method is the same as the method for synthesizing compound A having various R 1 and R 2 .
- P 3 represents a hydroxy protecting group, and other symbols are the same as defined above.
- the carbonyl compound E can be obtained by dihydroxylating the terminal olefin and performing oxidative cleavage with an oxidizing agent.
- an oxidizing agent for example, a method of reacting a catalytic amount of osmium tetroxide with sodium periodate in a solvent can be mentioned.
- Compound G Reduction of Carbonyl and Conversion to Leaving Group Carbonyl can be converted to hydroxy using a suitable reducing agent (eg, sodium borohydride).
- a suitable reducing agent eg, sodium borohydride
- Compound G can be synthesized by, for example, sulfonated the produced hydroxy (eg, methanesulfonate, p-toluenesulfonate). For example, it can be carried out by reacting chlorosulfonic acid chloride or p-toluenesulfonic acid chloride with an appropriate base (for example, triethylamine or N, N-dimethyl-4-aminopyridine).
- an appropriate base for example, triethylamine or N, N-dimethyl-4-aminopyridine.
- compound K can be synthesized by reacting with an appropriate base (for example, sodium hydride) in a solvent. Moreover, it may cyclize without adding a base.
- an appropriate base for example, sodium hydride
- the reaction is carried out after protecting the hydroxy located at the ⁇ -position of 2′-position and the amino group which may have a substituent, and then reacting with 2 ′ of Compound G A hydroxy- or amino-protected compound (compound G in which the 2′-position is protected) that may have a substituent is obtained, and the 2′-position of the compound G in which the 2′-position is protected is deprotected Then, a cyclization reaction may be performed.
- the process of obtaining compound K ′ from compound E via compound G ′ is the same.
- Compound M as a starting material can be synthesized according to methods known to those skilled in the art, such as the method described in Journal of the Chemical Society, Perkin Transaction 1, 1998, p 1409.
- the compound M having various R 1 , R 2 , R 3 , R 11 can be obtained from, for example, the following protection / deprotection reactions (for example, the Protective Reactions described in Groups in Organic Synthesis 4th edition), oxidation reactions, reduction reactions (for example, see Comprehensive Organic Transformations, 2nd edition, RCLarock, Wiley-VCH (1999) etc.) Can be synthesized.
- the specific method is the same as the method for synthesizing compound A having various R 1 and R 2 .
- the compound M in which at least one of R 3 and R 11 is alkyl can be synthesized by first oxidizing hydroxy and then reducing using an alkyl metal reagent or Grignard reagent.
- Dihydroxylation of Olefin Compound N can be synthesized by reacting an olefin at the 3 ′ position with an appropriate dihydroxylation reagent in a solvent. Dihydroxylation can be performed, for example, by using a catalytic amount of ruthenium chloride and a stoichiometric amount or more of sodium periodate.
- Alkylation of Primary Alcohol can be synthesized by reacting primary alcohol compound N with a suitable alkylating reagent in a solvent.
- the alkylation can be carried out, for example, by reacting with an alkyl halide in the presence of a suitable base (for example, N, N-diisopropylethylamine).
- Z 1 means a nucleotide structure represented by the following formula (Z 1 ).
- Z 2 means a nucleotide structure represented by the following formula (Z 2 ).
- Labeling with 6-carboxyfluorescein at the 5 ′ end is performed by removing a hydrogen atom from the hydroxy group at the 5 ′ end, Through a group represented by the formula —P ( ⁇ O) —O— (CH 2 ) 6 —N (H) —, a moiety obtained by removing a hydroxy group from one carboxy group of 6-carboxyfluorescein is bonded.
- a nitrogen atom is bonded to a portion obtained by removing a hydroxy group from one carboxy group of 6-carboxyfluorescein
- a phosphorus atom is bonded to a portion obtained by removing a hydrogen atom from the hydroxy group at the 5 ′ end.
- Example 1 Antisense oligonucleotides listed in Table 1 were prepared using an automated nucleic acid synthesizer.
- the target gene is mouse Superoxide Dismutase-1 (SOD-1). It has been reported that the antisense oligonucleotide of Comparative Example 1 having no modification in the gap region causes high toxicity due to the off-target effect (Nucleic Acids Research, 2016, 44, p 2093).
- the molecular weight of the synthesized oligonucleotide was measured by MALDI-TOF-MASS. The results are shown in Table 1.
- Mouse brain endothelial cell line bEND. 3 cells were suspended in DMEM medium containing 10% fetal bovine serum at 5000 cells / well, seeded in a 96-well plate (Corning Inc., # 3585) and about 37 ° C. under 5% CO 2. Cultured for 24 hours. Each oligonucleotide of Table 1 is dissolved in DMEM medium containing 10% fetal calf serum containing 10 mM calcium chloride (test medium) so that its final concentration is 10 nM, 30 nM, 100 nM, 300 nM or 1000 nM. After about 24 hours, the culture medium was replaced with a test medium (see Nucleic Acids Research, 2015, 43, pe128).
- Example 1 the antisense oligonucleotide according to the present invention exhibits the same antisense effect as the antisense oligonucleotide without any modification in the gap region (Comparative Example 1). It was done.
- Mouse brain endothelial cell line bEND. 3 cells are suspended in DMEM medium containing 10% fetal bovine serum so as to be 5000 cells / well, seeded in a 96-well plate (Corning, # 3585), and about 37 ° C. under 5% CO 2. Cultured for 24 hours.
- Each oligonucleotide of Table 1 is dissolved in DMEM medium containing 10% fetal calf serum containing 10 mM calcium chloride (test medium) so that its final concentration is 10 nM, 30 nM, 100 nM, 300 nM or 1000 nM.
- the culture medium was replaced with a test medium (see Nucleic Acids Research, 2015, 43, pe128). Further, 100 ⁇ L of ATP reagent (CellTiter-Glo TM Luminescent Cell Viability Assay, manufactured by Promega) was added to the cell culture solution after 7 days, suspended, and allowed to stand at room temperature for about 10 minutes, followed by FlexStation 3 (Molcular Devices). Luminescence intensity (RLU value) was measured, and the number of viable cells was measured by subtracting the luminescence value of the medium alone as an average value of three points. Cells to which no oligonucleotide was added were used as controls.
- ATP reagent CellTiter-Glo TM Luminescent Cell Viability Assay, manufactured by Promega
- Example 1 the antisense oligonucleotide according to the present invention has lower cytotoxicity than the antisense oligonucleotide without any modification in the gap region (Comparative Example 1). It was done. This result suggests that the cytotoxicity caused by the off-target effect seen in Comparative Example 1 is reduced by inserting a nucleic acid having a 2′-position and a 3′-position crosslinked in the gap region.
- RNA complementary to the oligonucleotides of Example 1 and Comparative Example 1 (RNA (SOD-1)) labeled with 6-carboxyfluorescein at the 5 ′ end was synthesized.
- the molecular weight of RNA (SOD-1) was measured by MALDI-TOF-MASS. The measured molecular weight was 5645.58 (M ⁇ H ⁇ ).
- coli-derived recombinant RNase H (manufactured by Wako Pure Chemical Industries, Ltd.) (1 unit) was added and reacted at 30 ° C. for 2 hours.
- the enzyme was inactivated by transferring to an oil bath at 90 ° C. and holding for 5 minutes, and RNA cleavage activity was measured by reverse phase HPLC.
- conversion rate indicates the rate at which RNA (16 mer) was cleaved, and is expressed as [100 ⁇ (RNA (16 mer) ⁇ (total of area values of each peak) ⁇ 100)].
- Numberers in bold letters (mer) represent cleaved RNA fragments, each represented by the number of nucleotides counted from the 5 ′ end.
- “Cleaved RNA area (%)” represents an area percentage (%) of each RNA fragment peak.
- the antisense oligonucleotide according to the present invention (Example 1) is closer to the modification position than the antisense oligonucleotide without any modification in the gap region (Comparative Example 1). It was shown that the selectivity of the cutting position was improved. From the results of Evaluation Examples 1 to 3, it was suggested that the modification that improves the selectivity of the cleavage position reduces the cytotoxicity.
- Example 2 Antisense oligonucleotides listed in Table 4 were prepared using an automated nucleic acid synthesizer.
- the target gene is mouse coagulation factor XI (FXI). It has been reported that the antisense oligonucleotide of Comparative Example 2 having no modification in the gap region causes high toxicity due to the off-target effect (Nucleic Acids Research, 2016, 44, pp 2093-2109).
- the molecular weight of the synthesized oligonucleotide was measured by MALDI-TOF-MASS. The results are shown in Table 4.
- Example 2 has lower cytotoxicity than the antisense oligonucleotide without any modification in the gap region (Comparative Example 2). It was done. This result suggests that the cytotoxicity caused by the off-target effect observed in Comparative Example 2 can be reduced by inserting a nucleic acid having a 2′-position and a 3′-position crosslinked in the gap region.
- RNA complementary to the oligonucleotides of Example 2 and Comparative Example 2 (RNA (FXI)) labeled with 6-carboxyfluorescein at the 5 ′ end was synthesized.
- the molecular weight of RNA (FXI) was measured by MALDI-TOF-MASS. The measured molecular weight was 5773.54 (M ⁇ H ⁇ ).
- RNA cleavage activity was measured using the same evaluation method as in Evaluation Example 3. However, the reaction time was 1.5 hours.
- Example 2 the antisense oligonucleotide according to the present invention was compared with the antisense oligonucleotide without any modification in the gap region (Comparative Example 2), in the region close to the modification position. It was shown that the selectivity of the cutting position was improved (8mer and 10mer production suppression). From the results of Evaluation Example 4 and Evaluation Example 5, it was suggested that the modification that improves the selectivity of the cleavage position reduces the cytotoxicity.
- Antisense oligonucleotides listed in Table 7 were prepared using an automated nucleic acid synthesizer.
- the target gene is human mutant Huntington (muHTT), which is a site having a SNP mutated from wild type A to G (see Molecular Therapy-Nucleic Acids, 2017, 7, pp 20-30).
- RNA (mu-HTT, fully complementary to antisense oligonucleotide) and wild-type RNA (wt-HTT, antisense oligonucleotide) labeled with 6-carboxyfluorescein at the 5 ′ end as described in Table 8 Containing a single base mismatch).
- the molecular weights of the above mu-HTT and wt-HTT were measured by MALDI-TOF-MASS. The measured molecular weight was as follows. mu-HTT: 5367.79 (M ⁇ H ⁇ ) wt-HTT: 5382.02 (M ⁇ H ⁇ )
- RNA fragments cleaved from mu-HTT 7-mer to 13-mer were separately prepared using an automatic nucleic acid synthesizer, and the molecular weight was measured by MALDI-TOF-MASS. The measured molecular weight was as follows.
- Mouse brain endothelial cell line bEND. 3 cells were suspended in DMEM medium containing 10% fetal bovine serum so as to be 40,000 cells / well, seeded in a 6-well plate (Corning, # 3516), and about 37 ° C. under 5% CO 2. Cultured for 24 hours. Each oligonucleotide in Table 1 was dissolved in DMEM medium containing 10% fetal calf serum containing 10 mM calcium chloride so that the final concentration was 3000 nM (test medium), and replaced with the test medium after about 24 hours. And cultured (see Nucleic Acids Research, 2015, 43, pe128).
- RNA fluorescently labeled with Cy3 [cyanine-3] was prepared from total RNA.
- Fluorescently labeled complementary RNA and SurePrint G3 Mouse Gene Expression 8x60K v2 were hybridized by the one-color protocol.
- the obtained signal data was analyzed using GeneSpring software (manufactured by Agilent Technologies), and the variation in gene expression relative to the control was comprehensively analyzed.
- the result of Comparative Example 1 is shown in FIG. 4, and the result of Example 1 is shown in FIG. 4 and 5, the horizontal axis (log 2 expression of control experiment) represents the expression level (log 2) in the control sample, and the vertical axis (log 2 fold change) represents the expression change relative to the control. Represents the ratio (log2).
- the antisense oligonucleotide without modification in the gap region is 760, whereas the anti-sense according to the present invention is The sense oligonucleotide (Example 1) was 564. From this, it was confirmed that the antisense oligonucleotide (Example 1) which concerns on this invention has suppressed the off-target effect.
- Example 4 Antisense oligonucleotides listed in Table 10 were prepared using an automated nucleic acid synthesizer.
- the target gene is mouse coagulation factor XI (FXI) as in Example 2 and Comparative Example 2.
- FXI mouse coagulation factor XI
- the molecular weight of the synthesized oligonucleotide was measured by MALDI-TOF-MASS. The results are shown in Table 10.
- the antisense oligonucleotide according to the present invention (Example 4) is closer to the modification position than the antisense oligonucleotide without modification in the gap region (Comparative Example 2). It was shown that the selectivity of the cutting position was improved (8mer and 10mer production suppression).
- the oligonucleotide of the present invention can suppress the off-target effect, it is considered that the toxicity can be reduced, such as metabolic diseases, tumors, infectious diseases, etc. It is useful as a pharmaceutical composition for treatment and prevention.
Abstract
Description
ギャップマー型アンチセンス核酸を臨床に応用するためには、高い配列特異性が求められる。しかしながら近年、オフターゲット効果に起因する毒性が報告されている(例えば、非特許文献3及び4参照)。オフターゲット効果は、アンチセンス核酸と標的以外の類似した配列を有するRNAとが二重鎖複合体を形成し、当該標的以外のRNAが切断されることによって生じる。しかしながら、このような毒性を低減する修飾方法に関する報告はない。
また、一塩基多型(SNP)を有する遺伝子を標的とした場合では、野生型に対する変異型の選択性が求められており、糖部をフッ素で修飾した人工核酸を用いた検討が報告されている(例えば、非特許文献5参照)。
また、一塩基多型(SNP)部位を標的とした場合では、野生型に対する変異型の選択性の向上が求められているが、ギャップマー型アンチセンス核酸の配列は、野生型RNAの配列と一塩基のミスマッチを含むのみであり、野生型/変異型の選択性を得るのは依然として難しい。よって、これを解決する新たな技術が求められている。
ここで、
- 中央領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群より独立して選択される少なくとも5個のヌクレオチドからなり、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から選択される糖部修飾ヌクレオチドを少なくとも一つ含み、その3’末端及び5’末端が、それぞれ独立して、デオキシリボヌクレオチド、リボヌクレオチド、2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドであり、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を、少なくとも一つ含み;
- 5’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その3’末端が、糖部修飾ヌクレオチドであり、ここで、該3’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まず;
- 3’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端が、糖部修飾ヌクレオチドであり、ここで、該5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない、
アンチセンスオリゴヌクレオチド。
前記5’側領域及び前記3’側領域が、それぞれ独立して、1~7個のヌクレオチドからなる、1.に記載のアンチセンスオリゴヌクレオチド。
前記5’側領域及び前記3’側領域が、それぞれ独立して、2~5個のヌクレオチドからなる、1.又は2.に記載のアンチセンスオリゴヌクレオチド。
下記式(I):
(式中、mは、1、2、3又は4であり、
Bxは、核酸塩基部分であり、
Xは、O又はSであり、
-Q-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であり、
mが、2、3又は4であるとき、2つの隣り合った-Q-は、一緒に
式 -CR7=CR8-
で表される基を形成してもよく、
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;
R6は、C1-C12アルキル又はアミノ保護基であり、
R7及びR8は、それぞれ独立して、水素原子又はC1-C6アルキルである)
で表される部分構造を含むヌクレオチドである、1.から3.の何れか一つに記載のアンチセンスオリゴヌクレオチド。
下記式(II):
(式中、Bxは、核酸塩基部分であり、
Xは、O又はSであり、
R12は、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
R1、R2、R3及びR11は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基である)
で表される部分構造を含むヌクレオチドである、1.から3.の何れか一つに記載のアンチセンスオリゴヌクレオチド。
下記式(III):
(式中、Bxは、核酸塩基部分であり、
Xは、O又はSであり、
-Q1-及び-Q2-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であるか、あるいは、
-Q1-Q2-は、-CR7=CR8-であり;かつ、式中、R7及びR8は独立して水素原子又はC1-C6アルキルであり、
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;
R6は、C1-C12アルキル又はアミノ保護基である)
で表されるヌクレオチドである、4.に記載のアンチセンスオリゴヌクレオチド。
前記5’側領域が、5’ウィング領域であり、
前記3’側領域が、3’ウィング領域である、1.から10.の何れか一つに記載のアンチセンスオリゴヌクレオチド。
(ii)少なくとも1個のリボヌクレオチドを含み、かつ前記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチド
を含む、オリゴヌクレオチド複合体。
(ii)少なくとも1個のリボヌクレオチドを含み、かつ前記(i)のアンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチドに由来する基
を含み、前記(i)アンチセンスオリゴヌクレオチドに由来する基と、前記(ii)オリゴヌクレオチドに由来する基とが連結された、オリゴヌクレオチド。
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチド
を含むオリゴヌクレオチド複合体であって、
前記(iii)の少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド複合体。
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチドに由来する基
を含み、前記(iii)オリゴヌクレオチドに由来する基と、前記(iv)オリゴヌクレオチドに由来する基とが連結され、
前記(iii)の少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド。
保護基により置換された官能基とは、官能基が有する水素原子が保護基により置換された官能基を意味する。
「C1-C6アルキル」とは、前記「C1-C12アルキル」のうち、炭素数が1から6の直鎖又は分枝状の飽和脂肪族炭化水素の1価の基を意味する。C1-C6アルキルとしては、例えば、メチル、エチル、n-プロピル、イソプロピル、n-ブチル、イソブチル、s-ブチル、t-ブチル、n-ペンチル、イソペンチル、ネオペンチル、n-ヘキシル、イソヘキシル等が挙げられる。同様に「C1-C3アルキル」とは、炭素数が1から3の直鎖又は分枝状の飽和脂肪族炭化水素の1価の基を意味する。
「ハロアシル」とは、前記「アシル」の任意の位置の水素原子の少なくとも1個が、前記「ハロゲン原子」で置換された基を意味する。
「C2-C20アルキレン」とは、炭素数が2から20の直鎖又は分枝状の飽和脂肪族炭化水素の2価の基を意味する。
「C8-C12アルキレン」とは、前記「C2-C20アルキレン」のうち、炭素数が8から12の直鎖又は分枝状の飽和脂肪族炭化水素の2価の基を意味する。
「C2-C6アルキレン」とは、前記「C2-C20アルキレン」のうち、炭素数が2から6の直鎖又は分岐状の飽和脂肪族炭化水素の2価の基を意味し、例としては、エチレン(エタンジイル)、プロピレン、プロパン-1,3-ジイル(トリメチレン)、プロパン-2,2-ジイル(イソプロピリデン)、2,2-ジメチル-プロパン-1,3-ジイル、ヘキサン-1,6-ジイル(ヘキサメチレン)及び3-メチルブタン-1,2-ジイル等が挙げられる。
「ジC1-C6アルキルアミノ」とは、アミノ(NH2)基の水素原子の2つが、同一又は異なる2つの前記「C1-C6アルキル」に置き換えられた基を意味し、例えば、ジメチルアミノ、ジエチルアミノ、ジn-プロピルアミノ、ジイソプロピルアミノ、ジn-ブチルアミノ、ジn-ペンチルアミノ、ジn-ヘキシルアミノ、N-メチル-N-エチルアミノ、及びN-メチル-N-イソプロピルアミノが挙げられる。
「チオキソ」とは、酸素原子が二重結合を介して置換した基(=S)を示す。チオキソが炭素原子に置換した場合は当該炭素原子と一緒となってチオカルボニルを形成する。
本発明の「アンチセンスオリゴヌクレオチド」分子が含むヌクレオチドは、それぞれ独立して、互いにホスホジエステル結合、後述する修飾されたホスホジエステル結合又は後述する非ヌクレオチド構造を含む連結基で連結されている。本発明のアンチセンスオリゴヌクレオチド分子の3’末端のヌクレオチドは、その3’位に、好ましくはヒドロキシ基又はリン酸基を有し、より好ましくはヒドロキシ基を有し、通常はヒドロキシ基を有する。アンチセンスオリゴヌクレオチド分子の5’末端のヌクレオチドは、その5’位に、好ましくはヒドロキシ基又はリン酸基を有し、より好ましくはヒドロキシ基を有し、通常はヒドロキシ基を有する。
前記置換と置き換えを組み合わせて、2’,4’-BNAの2’位と4’位を架橋する基は、-C(=O)-O-、-O-C(=O)-NR13-(R13は、水素原子、C1-C6アルキル又はハロC1-C6アルキルを示す)、-C(=O)-NR13-(R13は、水素原子、C1-C6アルキル又はハロC1-C6アルキルを示す)、-C(=S)-NR13-(R13は、水素原子、C1-C6アルキル又はハロC1-C6アルキルを示す)等で表される基を含んでいてもよい。
Bxは、核酸塩基部分であり、
Xは、O又はSであり、
-Q-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であり、
mが、2、3又は4であるとき、2つの隣り合った-Q-は、一緒に
式 -CR7=CR8-
で表される基を形成してもよく、
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;
R6は、C1-C12アルキル又はアミノ保護基であり、
R7及びR8は、それぞれ独立して、水素原子又はC1-C6アルキルである。
Xは、O又はSであり、
R12は、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
R1、R2、R3及びR11は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基である。
よって、DNAは、RNAとハイブリダイズした際に、RNaseHによって認識され得る。DNA及びRNAの少なくとも一方において、塩基部分、ホスホジエステル結合部分及び糖部分の少なくとも1つが修飾された場合も、同様である。例えば、代表的なものとして、DNAのホスホジエステル結合部分が、ホスホロチオエートに修飾されたオリゴヌクレオチド等が挙げられる。
RNAは、DNAとハイブリダイズした際に、RNaseHによって切断され得る。DNA及びRNAの少なくとも一方において、塩基部分、ホスホジエステル結合部分及び糖部分の少なくとも1つが修飾された場合も、同様である。
RNaseHによって認識され得るDNA及び/又はRNAの修飾例は、例えば、Nucleic Acids Research, 2014, 42, pp 5378-5389、Bioorganic & Medicinal Chemistry Letters, 2008, 18, pp 2296-2300(上述の、非特許文献1)、Molecular BioSystems, 2009, 5, pp 838-843、Nucleic Acid Therapeutics, 2015, 25, pp 266-274、The Journal of Biological Chemistry, 2004, 279, pp 36317-36326(上述の、非特許文献2)等に記載されている。
本発明に用いられるRNaseHは、好ましくは哺乳動物のRNaseHであり、より好ましくはヒトのRNaseHであり、特に好ましくはヒトRNaseH1である。
「5’側領域」は、前記「中央領域」の5’側に連結し、少なくとも1個のヌクレオチドを含む領域である。
「3’側領域」は、前記「中央領域」の3’側に連結し、少なくとも1個のヌクレオチドを含む領域である。
ある少なくとも4個の連続するヌクレオチドが、「RNaseHによって認識される少なくとも4個の連続するヌクレオチド」であるかどうか、当業者は、その連続するヌクレオチドの糖部分の構造により、判断できる。
本発明のアンチセンスオリゴヌクレオチドは、標的RNAの全体とハイブリダイズする必要はなく、標的RNAの少なくとも一部とハイブリダイズすればよいが、通常は、標的RNAの少なくとも一部とハイブリダイズする。例えば、標的RNAの部分配列に相補的なアンチセンス配列を有するオリゴヌクレオチド(DNA、オリゴデオキシリボヌクレオチド又は通常アンチセンス効果が生じるように設計されたオリゴヌクレオチド等)と標的RNAの少なくとも一部がハイブリダイズすることによって、標的遺伝子の発現が制御される。また、アンチセンスオリゴヌクレオチドの全体がハイブリダイズする必要はなく、一部がハイブリダイズしなくてもよい。アンチセンス配列部分の全体は、一部がハイブリダイズしなくてもよいが、ハイブリダイズすることが、好ましい。
中央領域は、デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群より独立して選択される少なくとも5個のヌクレオチドからなり、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から選択される糖部修飾ヌクレオチドを少なくとも一つ含み、その3’末端及び5’末端が、それぞれ独立して、デオキシリボヌクレオチド、リボヌクレオチド、2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドであり、そしてデオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を、少なくとも一つ含む。
核酸塩基部分には、前述の「核酸塩基」を用いることができる。
mは、1、2、3又は4である。
-Q-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であり、mが、2、3又は4であるとき、2つの隣り合った-Q-は、一緒に
式 -CR7=CR8-
で表される基を形成してもよい。
R6は、好ましくはC1-C3アルキルであり、より好ましくはメチルである。
mが2の場合、中央領域に含まれる、好ましい2’-3’架橋ヌクレオチドの部分構造は、下記式(III)で示される。
核酸塩基部分には、前述の「核酸塩基」を用いることができる。
Xは、O又はSである。
-Q1-及び-Q2-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であるか、又は、-Q1-Q2-は、-CR7=CR8-であり;かつ、式中、R7及びR8は独立して水素原子又はC1-C6アルキルである。
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;R6は、C1-C12アルキル又はアミノ保護基である。
-Q1-が-O-、-NH-、-NR6-又は-S-であり、該R6はC1-C12アルキルであり、-Q2-が-CH2-であることが好ましく、-Q1-が-O-であり、-Q2-が-CH2-であることがさらに好ましい。
Xは、O又はSである。
R12は、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択される。
R1、R2、R3及びR11は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基である。
R11は、好ましくは水素原子又はC1-C3アルキルであり、より好ましくは水素原子である。
R12は、好ましくはC1-C6アルキル又は1つ以上の置換基で置換されたC1-C6アルキルであり、より好ましくはC1-C3アルキルであり、特に好ましくはメチルである。
中央領域に含まれる2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドは、中央領域の任意の箇所に含むことができるが、中央領域の3’末端から数えて3つ目のヌクレオチドから、5’末端までの間に含むことが好ましい。2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドを含む箇所は、通常、前記標的RNAに対するアンチセンス効果の強さ、毒性の低さ等の他の要素に応じて選択される。
SNPを有する部分を標的とする場合、ある実施態様においては、変異した塩基と塩基対を形成する塩基と近い配列部分(例えば、変異した塩基と塩基対を形成する塩基から数えて、5つ目以内、4つ目以内、3つ目以内、2つ目以内、1つ目以内)に、2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドを含むことが好ましい。変異した塩基と塩基対を形成する塩基が、2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドであることが特に好ましい。
3’側領域は、デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端が、糖部修飾ヌクレオチドであり、ここで、該5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そしてデオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない。
2’位修飾非架橋ヌクレオチドは、好ましくは、2’-O-メチル化ヌクレオチド、2’-O-メトキシエチル(MOE)化ヌクレオチド、2’-O-アミノプロピル(AP)化ヌクレオチド、2’-フルオロ化ヌクレオチド、2’-O-(N-メチルアセトアミド)(NMA)化ヌクレオチド及び2’-O-メチルカルバモイルエチル(MCE)化ヌクレオチドからなる群から独立して選択され、より好ましくは、2’-O-メトキシエチル(MOE)化ヌクレオチド及び2’-O-メチルカルバモイルエチル(MCE)化ヌクレオチドから独立して選択され、特に好ましくは、2’-O-メトキシエチル(MOE)化ヌクレオチドである。
2’,4’-BNAは、好ましくは、LNA、cEt-BNA、ENA、BNANC、AmNA及びscpBNAであり、より好ましくは下記式(VI)で表わされる部分構造を含むLNAである。3’側領域は、5’側領域と同様である。
別の好ましい形態として、5個の2’位修飾非架橋ヌクレオチドからなるオリゴヌクレオチドである。
別の好ましい形態として、5’側領域は、2’,4’-BNA及びデオキシリボヌクレオチドからなる群から独立して選択される2~5個のヌクレオチドからなり、少なくとも2個の2’,4’-BNAを含むオリゴヌクレオチドであり、そのようなオリゴヌクレオチドは国際公開第2016/127002号等を参考にできる。3’側領域は、5’側領域と同様である。
前記リンカー又は機能性分子と、アンチセンスオリゴヌクレオチド分子の末端ヌクレオチドとは、ホスホジエステル結合又は修飾されたホスホジエステル結合で連結されていることが好ましく、ホスホジエステル結合で連結されていることがより好ましい。
前記リンカー又は機能性分子は、アンチセンスオリゴヌクレオチド分子の3’末端のヌクレオチドが有する3’位の酸素原子又は5’末端のヌクレオチドが有する5’位の酸素原子に直接連結されていてもよい。
(式中eは、それぞれ独立して、1から6の整数である)で表される基であり、特に好ましくは、下記式:
で表される基である。
プロドラッグとは、化学的又は代謝的に分解できる基を有する医薬品化合物の誘導体であり、加溶媒分解又は生理的条件下のインビボ(in vivo)での分解により、薬理学的に活性な医薬品化合物へと誘導される化合物である。適当なプロドラッグ誘導体を選択する方法及び製造する方法は、例えばDesign of Prodrugs(Elsevier, Amsterdam, 1985)に記載されている。本発明の場合、ヒドロキシ基を有する場合は、その化合物と適当なアシルハライド、適当な酸無水物又は適当なハロゲン化アルキルオキシカルボニル化合物とを反応させることによって製造されるアシルオキシ誘導体のようなプロドラッグが例示される。プロドラッグとして特に好ましい構造としては、-O-C(=O)C2H5、-O-C(=O)(t-Bu)、-O-C(=O)C15H31、-O-C(=O) - (m-CO2Na-Ph)、-O-C(=O)CH2CH2CO2Na-OC(=O)CH(NH2)CH3、-O-C(=O)CH2N(CH3)2又は-O-CH2OC(=O)CH3などが挙げられる。本発明を形成するアンチセンスオリゴヌクレオチドがアミノ基を有する場合は、アミノ基を有する化合物と適当な酸ハロゲン化物、適当な混合酸無水物又は適当なハロゲン化アルキルオキシカルボニル化合物とを反応させることにより製造されるプロドラッグが例示される。プロドラッグとして特に好ましい構造としては、-NH-C(=O) - (CH2)20OCH3、-NH-C(=O)CH(NH2)CH3、-NH-CH2OC(=O)CH3等が挙げられる。
(i)前記アンチセンスオリゴヌクレオチド、及び
(ii)少なくとも1個のリボヌクレオチドを含み、かつ前記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチド
を含む、オリゴヌクレオチド複合体。
(i)前記アンチセンスオリゴヌクレオチドに由来する基、及び
(ii)少なくとも1個のリボヌクレオチドを含み、かつ上記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチドに由来する基
を含み、前記(i)アンチセンスオリゴヌクレオチドに由来する基と、前記(ii)のオリゴヌクレオチドに由来する基とが
連結されたオリゴヌクレオチド。
(iii)前記アンチセンスオリゴヌクレオチドに由来する基に、少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖が連結されたオリゴヌクレオチド、及び
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチド
を含むオリゴヌクレオチド複合体であって、
前記(iii)の少なくとも1個のリボヌクレオチドを含む前記オリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含む前記オリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド複合体。
(iii)前記アンチセンスオリゴヌクレオチドに由来する基に、少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖が連結されたオリゴヌクレオチドに由来する基、及び
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチドに由来する基
を含み、前記(iii)オリゴヌクレオチドに由来する基と、前記(iv)オリゴヌクレオチドに由来する基とが連結され、
前記(iii)の少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド。
生理的条件で分解されるオリゴヌクレオチドは、生理的条件で分解されるオリゴヌクレオチド内において部分的に相補的な配列を含んでいてもよく、含んでいなくてもよいが、好ましくは、部分的に相補的な配列を含まない。そのようなオリゴヌクレオチドの例として、ホスホジエステル結合で連結された(N)k’(Nは、それぞれ独立してアデノシン、ウリジン、シチジン、グアノシン、2’-デオキシアデノシン、チミジン、2’-デオキシシチジン、または2’-デオキシグアノシンであり、kは1~40の整数(繰り返し数)である)を挙げることができる。中でも、k’は好ましくは3~20であり、より好ましくは4~10であり、さらに好ましくは4~7であり、さらにより好ましくは、4又は5であり、特に好ましくは4である。
{式中、V11は、
C2-C50アルキレン
(該C2-C50アルキレンは、無置換であるか又は、置換基群Vaより独立して選択される1つ以上の置換基で置換されている)、
下記式(XIII-1)から(XIII-11):
からなる群より選択される基、
リボヌクレオシド基、又は
デオキシリボヌクレオシド基であり、
少なくとも1つのV11は、C2-C50アルキレン(該C2-C50アルキレンは、無置換であるか、又は置換基群Vaより独立して選択される1つ以上の置換基で置換されている)、又は前記式(XIII-1)から(XIII-11)より選択される基であり、
置換基群Vaは、ヒドロキシ、ハロゲン原子、シアノ、ニトロ、アミノ、カルボキシ、カルバモイル、スルファモイル、ホスホノ、スルホ、テトラゾリル及びホルミルにより構成される置換基群を意味し、
P11は、それぞれ独立して、-P(=O)(OH)- 又は -P(=O)(SH)- であり、
少なくとも1つのP11は、-P(=O)(OH)- であり、
q11は、1から10の整数であり、q12は、1から20の整数であり、q11及びq12の少なくとも一方が2以上のとき、V11は、同一であるか又は異なっている。}
糖部修飾ヌクレオチド、デオキシリボヌクレオチド及びリボヌクレオチドの種類、数及び位置は、ここで開示するアンチセンスオリゴヌクレオチドのプロドラッグが奏するアンチセンス効果等に影響を与えうる。その種類、数及び位置は、標的RNAの配列等によっても異なるため、一概には言えないが、当業者は、前記アンチセンス法に関する文献の記載を参酌しながら、好ましい態様を決定することができる。また、塩基部分、糖部分又はホスホジエステル結合部分の修飾後のアンチセンスオリゴヌクレオチドのプロドラッグが有するアンチセンス効果を測定し、得られた測定値が、修飾前のアンチセンスオリゴヌクレオチドのプロドラッグのそれよりも有意に低下していなければ(例えば、修飾後アンチセンスオリゴヌクレオチドのプロドラッグの測定値が修飾前のプロドラッグの測定値の30%以上であれば)、当該修飾が好ましい態様であると評価することができる。アンチセンス効果の測定は、例えば、後述の実施例において示されているように、細胞等に被検オリゴヌクレオチドを導入し、該被検オリゴヌクレオチドが奏するアンチセンス効果によって制御された標的RNAの発現量、標的RNAに関連しているcDNAの発現量、標的RNAに関連しているタンパク質の量等を、ノザンブロッティング、定量的PCR、ウェスタンブロッティング等の公知の手法を適宜利用することによって行うことができる。
その他の態様として、(ii)のオリゴヌクレオチド又はオリゴヌクレオチドに由来する基は、リボヌクレオチド及び糖部修飾ヌクレオチドから選択され、当該糖部修飾ヌクレオチドは、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択される。このとき、当該オリゴヌクレオチドの末端が、少なくとも1つの糖部修飾ヌクレオチドであることが好ましい。この糖部修飾ヌクレオチドは、好ましくは、2’-O-メチル化ヌクレオチドであり、ホスホロチオエート結合で隣接するヌクレオチドと結合していることが好ましい。前記(C)に示されるオリゴヌクレオチド複合体又は(D)に示されるオリゴヌクレオチドにおいて、(iii)のオリゴヌクレオチドのうち少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖(RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖とハイブリダイズする部分も同様である。
上記(i)アンチセンスオリゴヌクレオチドと、(ii)少なくとも1個のリボヌクレオチドを含み、かつ上記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域とがハイブリダイズした部分が、RNaseHによって認識され、(ii)少なくとも1個のリボヌクレオチドを含み、かつ上記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域が切断されると考えられる。その結果、標的細胞等において、本発明のアンチセンスオリゴヌクレオチドが生成し、(A)のプロドラッグは治療効果等を発揮すると考えられる。前記(B)についても同様である。
前記(C)においては、
前記(iii)の少なくとも1個のリボヌクレオチドを含む前記オリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含む前記オリゴヌクレオチド鎖とが、ハイブリダイズした部分が、RNaseHによって認識され、(iii)の少なくとも1個のリボヌクレオチドを含む前記オリゴヌクレオチド鎖が切断されると考えられる。その結果、標的細胞等において、本発明のアンチセンスオリゴヌクレオチドが生成し、(C)のプロドラッグは治療効果等を発揮すると考えられる。前記(D)についても同様である。
前記(C)及び(D)のうち、(iv)のオリゴヌクレオチド又はオリゴヌクレオチドに由来する基は、アンチセンスオリゴヌクレオチド又はアンチセンスオリゴヌクレオチドに由来する基であってもよい。当該(iv)のアンチセンスオリゴヌクレオチド又はアンチセンスオリゴヌクレオチドに由来する基は、(iii)のオリゴヌクレオチドが含むアンチセンスオリゴヌクレオチドに由来する基と同一でもよく、異なっていてもよい。また、本発明のアンチセンスオリゴヌクレオチドに由来する基であってもなくてもよい。前記(iv)のアンチセンスオリゴヌクレオチド又はアンチセンスオリゴヌクレオチドに由来する基は、(iii)のアンチセンスオリゴヌクレオチド又はアンチセンスオリゴヌクレオチドに由来する基とは、ハイブリダイズしないことが好ましい。
本発明のアンチセンスオリゴヌクレオチドでないアンチセンスオリゴヌクレオチドとしては、例えば、以下のアンチセンスオリゴヌクレオチドが挙げられる。
- 中央領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群より独立して選択される少なくとも5個のヌクレオチドからなり、前記糖部修飾ヌクレオチドは、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、
その3’末端及び5’末端が、それぞれ独立して、デオキシリボヌクレオチド又はリボヌクレオチドであり、そして
デオキシリボヌクレオチドから独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を、少なくとも一つ含み;
- 5’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その3’末端が、糖部修飾ヌクレオチドであり、ここで、該3’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まず;
- 3’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端が、糖部修飾ヌクレオチドであり、ここで、該5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない、
アンチセンスオリゴヌクレオチド。
(2)中央領域、5’側領域及び3’側領域を有するアンチセンスオリゴヌクレオチドであって、ここで、
- 中央領域は、
デオキシリボヌクレオチドから独立して選択される少なくとも5個のヌクレオチドからなり、
- 5’側領域は、
デオキシリボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その3’末端が、糖部修飾ヌクレオチドであり、ここで、該3’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まず;
- 3’側領域は、
デオキシリボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端が、糖部修飾ヌクレオチドであり、ここで、該5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない、
アンチセンスオリゴヌクレオチド。
(3)中央領域、5’側領域及び3’側領域を有するアンチセンスオリゴヌクレオチドであって、ここで、
- 中央領域は、
デオキシリボヌクレオチドから独立して選択される少なくとも5個のヌクレオチドからなり、
- 5’側領域は、
糖部修飾ヌクレオチドから独立して選択される少なくとも1個のヌクレオチドからなり、その3’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、
- 3’側領域は、
デオキシリボヌクレオチドから独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択される、
アンチセンスオリゴヌクレオチド。
(4)デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群より独立して選択される少なくとも5個のヌクレオチドからなり、
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない、
アンチセンスオリゴヌクレオチド。
特に、-P(=O)(OH)-で表される部分構造が、-P(=O)(O-)-で表されるアニオン性の部分構造へ変換されて、アルカリ金属(リチウム、ナトリウム、カリウムなど)、アルカリ土類金属(マグネシウム、カルシウムなど)又はアンモニウム等と塩を形成してもよい。また、ホスホロチオエート結合を形成する、-P(=O)(SH)-で表される部分構造が、-P(=O)(S-)-で表されるアニオン性の部分構造へ変換されて、同様に、アルカリ金属、アルカリ土類金属又はアンモニウム等と塩を形成しても
よい。その他の修飾されたホスホジエステル結合についても同様である。
本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグと、細胞を接触させる工程を含む、標的RNAの機能を制御する方法。
本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグを含む医薬組成物を、哺乳動物に投与する工程を含む、該哺乳動物おける標的RNAの機能を制御する方法。
哺乳動物において、標的RNAの機能を制御するための、本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグの使用。
哺乳動物において、標的RNAの機能を制御するための薬剤を製造するための、本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグの使用。
本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグと細胞を接触させる工程を含む、標的遺伝子の発現を制御する方法。
本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグを含む医薬組成物を、哺乳動物に投与する工程を含む、該哺乳動物おける標的遺伝子の発現を制御する方法。
哺乳動物において、標的遺伝子の発現を制御するための、本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグの使用。
哺乳動物において、標的遺伝子の発現を制御するための薬剤を製造するための、本発明のアンチセンスオリゴヌクレオチド又はそのプロドラッグの使用。
投与経路は、好ましくは経腸管的である。その他の態様として、投与経路は、非経腸管的である。
式中、P1及びP2は、それぞれ独立してヒドロキシ保護基であり、LG1は脱離基を表し、-Q-は、-CR4R5-、-C(=O)-、-C(=S)-、又は-C(=NR6)-を表し、R4、R5、R6、及びその他の記号は前記定義に同じである。
式中、P3は、ヒドロキシ保護基を表し、その他の記号は前記定義に同じである。
適当な塩基(例えば、DBU又は安息香酸ナトリウム)を用いて、脱離基を脱離させることによりオレフィン化体(化合物B)を得ることができる。例えば、溶媒中、安息香酸ナトリウムを反応させる方法が挙げられる。
-Q-が-CR4R5-である場合、通常知られているシクロプロパン化反応により環化体(C)を合成できる。例えば、溶媒中、アルキルで置換されていてもよいジヨードメタンを、ジエチル亜鉛と共に反応させる方法が挙げられる。
-Q-が-C(=O)-である場合、例えば、保護されたヒドロキシジヨードメタンを、ジエチル亜鉛と共に反応させ、その後保護されたヒドロキシを脱保護し、酸化する方法により、環化体(C)を合成できる。-Q-が-C(=S)-である場合、上記-C(=O)-である化合物に対して、ローソン試薬等でチオカルボニル化することにより、また、-Q-が-C(=NR6)-である場合、-Q-が-C(=O)-である前記化合物に対し、対応するアミノ基を有するアミンを用いたイミノ化により、環化体(化合物C)を合成できる。
式中、P1及びP2はヒドロキシ保護基であり、LG1及びLG2はそれぞれ独立して脱離基であり、Q11は、O、NH又はNR6であり、Hは水素原子であり、kは0~3の整数であり、R6及びその他の記号は前記定義に同じである。
多様なR1、R2を有する化合物Dは、例えば、以下に記載の化合物D-1から、当業者に公知の保護・脱保護反応(例えば、前記Protective Groups in Organic Synthesis 第4版に記載されている反応)、酸化反応、還元反応(例えば、Comprehensive Organic Transformations, 第2版、R.C.Larock著、Wiley-VCH(1999年)等を参照できる)を組み合わせて用い、合成することができる。具体的な方法は、多様なR1、R2を有する化合物Aの合成法と同じである。
式中、P3はヒドロキシ保護基を表し、その他の記号は前記定義に同じである。
2’位の脱離基に対し、溶媒中、例えば水酸化ナトリウム水溶液などの塩基と反応することで、ヒドロキシが2’位のβ位に位置したヒドロキシ体を得ることができる。2’位の脱離基に対し、溶媒中、置換基を有してもよいアミン又はアンモニアを反応させることで、置換基を有してもよいアミノ基が2’位のβ位に位置したアミノ体を得ることができる。又は、アジ化ナトリウムによる還元によってもアミノ体を得ることができる。
さらに、末端オレフィンをジヒドロキシル化し、酸化剤で酸化的開裂を行うことで、カルボニル化合物Eを得ることができる。例えば、溶媒中、触媒量の四酸化オスミウムと過ヨウ素酸ナトリウムを反応させる方法が挙げられる。
適当な還元剤(例えば、水素化ホウ素ナトリウム)を用いて、カルボニルをヒドロキシに変換できる。生成したヒドロキシを、例えばスルホネート化(例えば、メタンスルホネート化、p-トルエンスルホネート化)することで化合物Gを合成できる。例えば、塩化メタンスルホン酸又は塩化p-トルエンスルホン酸を、適当な塩基(例えば、トリエチルアミン又はN,N-ジメチル-4-アミノピリジン)と共に反応させることで実施できる。
例えば、溶媒中、適当な塩基(例えば、水素化ナトリウム)と反応させることで、化合物Kを合成できる。また、塩基を加えなくても、環化する場合がある。
例えば、溶媒中、適当な酸化剤(例えば、亜塩素酸)、リン酸二水素ナトリウム及び2-メチル-2-ブテンと反応することで、カルボン酸化合物G’を得ることができる。
(化合物K’の合成):環化
化合物G’のカルボキシと、ヒドロキシ又はアミノとを既知の方法により縮合することで、化合物K’を合成できる。また、カルボキシをエステル、活性エステル(N-ヒドロキシスクシンイミド化等)、酸クロリド等へ変換した後、既知の縮合反応により合成することもできる。
式中、P1はヒドロキシ保護基であり、その他の記号は前記定義に同じである。
多様なR1、R2、R3、R11を有する化合物Mは、例えば、以下に記載の化合物M-1又はM-2から、当業者に公知の保護・脱保護反応(例えば、前記Protective Groups in Organic Synthesis 第4版に記載されている反応)、酸化反応、還元反応(例えば、Comprehensive Organic Transformations, 第2版、R.C.Larock著、Wiley-VCH(1999年)等を参照できる)を組み合わせて用い、合成することができる。具体的な方法は、多様なR1、R2を有する化合物Aの合成法と同じである。
3’位のオレフィンに対し、溶媒中、適当なジヒドロキシ化試薬を用いて反応することで、化合物Nを合成できる。ジヒドロキシ化は、例えば、触媒量の塩化ルテニウムと化学量論量以上の過ヨウ素酸ナトリウムを用いることで実施できる。
一級のアルコール化合物Nに対して、溶媒中、適当なアルキル化試薬を用いて反応することで、化合物Sを合成できる。アルキル化は、例えば、適当な塩基(例えば、N、N-ジイソプロピルエチルアミン)存在下で、ハロゲン化アルキルと反応させることで実施できる。
化合物Uは、化合物Mのエポキシ化、得られたエポキシ化合物(化合物T)の還元によって合成できる。合成法は、Journal of the Chemical Society, Perkin Transaction 1, 1998, p 1409に記載の方法等を参照できる。
核酸自動合成機は、特に記載がない場合は、nS-8II(ジーンデザイン社製)を用いた。
実施例中の配列表記(表1、2、4、5、7、8、10)において、特に記載がない限り、「(L)」はLNAを意味し、小文字のアルファベットはデオキシリボヌクレオチドを意味し、大文字のアルファベット(前記(L)付のアルファベットは除く)はリボヌクレオチドを意味し、「^」はホスホロチオエート結合を意味し、「5」は、そのヌクレオチドの塩基が5-メチルシトシンであることを意味し、「(m)」は2’-O-MOE化ヌクレオチドを意味し、「FAM-」は、5’末端が6-カルボキシフルオレセインで標識されていることを意味する。また、Z1は下記式(Z1)で表されるヌクレオチド構造を意味する。
式 -P(=O)-O-(CH2)6-N(H)-で表される基を介して、6-カルボキシフルオレセインの1つのカルボキシ基からヒドロキシ基を取り除いた部分が、結合していることを意味する。なお、式中、窒素原子が6-カルボキシフルオレセインの1つのカルボキシ基からヒドロキシ基を取り除いた部分に結合し、リン原子は、5’末端のヒドロキシ基から水素原子を取り除いた部分に結合する。
2’-O-3’-C-架橋の修飾ヌクレオチドである、(1R,2R,4R,5S)-1-(2-シアノエトキシ(ジイソプロピルアミノ)ホスフィノキシ)-2-(4,4’-ジメトキシトリチルオキシメチル)-4-(チミン-1-イル)-3,6-ジオキサビシクロ-[3.2.0]ヘプタンは、Journal of the American Chemical Society, 1998, 120, pp 5458-5463に記載の方法に準じて合成した。
表1に記載されたアンチセンスオリゴヌクレオチドを、核酸自動合成機を使用して調製した。標的遺伝子は、マウスSuperoxide Dismutase-1(SOD-1)である。ギャップ領域に修飾が無い比較例1のアンチセンスオリゴヌクレオチドは、オフターゲット効果に起因する高い毒性を生じることが報告されている(Nucleic Acids Research, 2016, 44, p 2093)。
合成したオリゴヌクレオチドの分子量は、MALDI-TOF-MASSにより測定した。結果を表1に示す。
マウス脳内皮細胞株bEND.3の細胞を5000細胞/ウェルとなるように10%ウシ胎児血清を含んだDMEM培地に懸濁し、96ウェルプレート(コーニング社製、♯3585)に播き、5%CO2下37℃にて約24時間培養した。表1の各オリゴヌクレオチドを、その最終濃度が10nM、30nM、100nM、300nM又は1000nMとなるように10mMの塩化カルシウムを含んだ10%ウシ胎児血清を含んだDMEM培地に溶解し(試験培地)、約24時間後に試験培地に交換し培養した(Nucleic Acids Research, 2015, 43, p e128参照)。さらに7日後に細胞を回収し、細胞からRNeasy mini kit(QIAGEN社製)を用いてTotal RNAを抽出した。
Total RNAからPrimeScript RT Master Mix(タカラバイオ(株)製)を用いてcDNAを得た。得られたcDNA及びTaqMan(登録商標)Gene Expression ID(Applied Biosystems社製)を用いて7500 Real-Time PCR System(Applied Biosystems社製)によりリアルタイムPCRを行い、PTENのmRNA量を定量した。リアルタイムPCRでは、ハウスキーピング遺伝子のcyclophilinのmRNA量も同時に定量し、cyclophilinのmRNA量に対するPTENのmRNA量をPTEN発現レベルとして評価した。オリゴヌクレオチドの添加を行わなかった細胞をコントロールとして用いた。結果を図1に示す。
なお、用いたプライマーは、TaqMan Gene Expression Assay(Applied Biosystems社製)であり、Assay IDは、以下の通りであった:
マウスSOD-1定量用: Mm01344233_g1
マウスcyclophilin定量用: Mm0234230_g1
マウス脳内皮細胞株bEND.3の細胞を5000細胞/ウェルとなるように10%ウシ胎児血清を含んだDMEM培地に懸濁し、96ウェルプレート(コーニング社製、#3585)に播き、5%CO2下37℃にて約24時間培養した。表1の各オリゴヌクレオチドを、その最終濃度が10nM、30nM、100nM、300nM又は1000nMとなるように10mMの塩化カルシウムを含んだ10%ウシ胎児血清を含んだDMEM培地に溶解し(試験培地)、約24時間後に試験培地に交換し培養した(Nucleic Acids Research, 2015, 43, p e128参照)。さらに7日後の細胞培養液に対してATP試薬100μL(CellTiter-GloTM Luminescent Cell Viability Assay, Promega社製)を添加し懸濁させ、約10分間室温で静置下後、FlexStation 3(Molcular Devices社製)にて発光強度(RLU値)を測定し、培地のみの発光値を差し引き3点の平均値として生細胞の数を測定した。オリゴヌクレオチドの添加を行わなかった細胞をコントロールとして用いた。
表2に記載された、5’末端が6-カルボキシフルオレセインで標識された、実施例1および比較例1のオリゴヌクレオチドに相補なRNA(RNA(SOD-1))を合成した。RNA(SOD-1)の分子量は、MALDI-TOF-MASSにより測定した。分子量実測値は、5645.58(M-H-)であった。
溶離液:0.1Mのヘキサフルオロイソプロピルアルコールと8mMのトリエチルアミンとを含む水溶液/メタノール=95/5(1分)→(14分)→75/25(3.5分)
流速:1.0mL/min
カラム:WatersXBridgeTM C18 2.5μm,4.6mm×75mm
カラム温度:60℃
検出:蛍光(Em518nm、Ex494nm)
11mer: 4095.60(M-H-)
10mer: 3764.68(M-H-)
9mer: 3420.65(M-H-)
8mer: 3074.77(M-H-)
7mer: 2746.23(M-H-)
上記HPLC分析条件1でのそれらのピークの保持時間を確認した。その他の表3中のRNA断片は、上記HPLC分析条件1におけるピークの保持時間から推定できる。
表4に記載されたアンチセンスオリゴヌクレオチドを、核酸自動合成機を使用して調製した。標的遺伝子は、マウスcoagulation factor XI(FXI)である。ギャップ領域に修飾が無い比較例2のアンチセンスオリゴヌクレオチドは、オフターゲット効果に起因する高い毒性を生じることが報告されている(Nucleic Acids Research, 2016, 44, pp 2093-2109)。
合成したオリゴヌクレオチドの分子量は、MALDI-TOF-MASSにより測定した。結果を表4に示す。
評価例2と同じ評価方法を用いて、表4の各オリゴヌクレオチドの最終濃度が10nM、30nM、100nM、300nM又は1000nMとなるようにし、生細胞の数を測定した。オリゴヌクレオチドの添加を行わなかった細胞をコントロールとして用いた。
結果を図3に示す。
表5に記載された、5’末端が6-カルボキシフルオレセインで標識された、実施例2および比較例2のオリゴヌクレオチドに相補なRNA(RNA(FXI))を合成した。RNA(FXI)の分子量は、MALDI-TOF-MASSにより測定した。分子量実測値は、5773.54(M-H-)であった。
10mer: 3828.08(M-H-)
9mer: 3521.67(M-H-)
8mer: 3177.46(M-H-)
7mer: 2873.46(M-H-)
6mer: 2543.94(M-H-)
上記HPLC分析条件1でのそれらのピークの保持時間を確認した。その他の表3中のRNA断片は、上記HPLC分析条件1におけるピークの保持時間から推定できる。
表7に記載されたアンチセンスオリゴヌクレオチドを、核酸自動合成機を使用して調製した。標的遺伝子は、ヒト変異型ハンチントン(muHTT)であり、野生型のAからGに変異したSNPを有した箇所である(Molecular Therapy - Nucleic Acids, 2017, 7, pp 20-30参照)。
mu-HTT: 5367.79(M-H-)
wt-HTT: 5382.02(M-H-)
表7の各オリゴヌクレオチドと、表8の各RNAを用い、評価例3と同じ評価方法を用いて、RNAの切断活性を測定した。ただし、反応時間は0.5時間とした。
13mer: 4730.56(M-H-)
12mer: 4425.41(M-H-)
11mer: 4117.98(M-H-)
10mer: 3788.92(M-H-)
9mer: 3460.61(M-H-)
8mer: 3154.41(M-H-)
7mer: 2825.87(M-H-)
上記HPLC分析条件1でのそれらのピークの保持時間を確認した。その他の表9中のRNA断片は、上記HPLC分析条件1におけるピークの保持時間から推定できる。
マウス脳内皮細胞株bEND.3の細胞を40000細胞/ウェルとなるように10%ウシ胎児血清を含んだDMEM培地に懸濁し、6ウェルプレート(コーニング社製、#3516)に播き、5%CO2下37℃にて約24時間培養した。表1の各オリゴヌクレオチドを、その最終濃度が3000nMとなるように10mMの塩化カルシウムを含んだ10%ウシ胎児血清を含んだDMEM培地に溶解し(試験培地)、約24時間後に試験培地に交換し培養した(Nucleic Acids Research, 2015, 43, p e128参照)。さらに24時間後に細胞を回収し、細胞からRNeasy mini kit(QIAGEN社製)を用いてTotal RNAを抽出した。オリゴヌクレオチドの添加を行わなかった細胞をコントロールとして用いた。
常法に従い、Total RNAから Cy3[シアニン-3]で蛍光標識した相補的RNAを作製した。蛍光標識した相補的RNAと、SurePrint G3 Mouse Gene Expression 8x60K v2(Agilent Technologies社製)とを、一色法(one-color protocol)にてハイブリダイズさせた。得られたシグナルデータは、GeneSpring ソフトウエア(Agilent Technologies社製)を用いて分析し、コントロールに対する遺伝子の発現量の変動を網羅的に解析した。比較例1の結果を図4に、実施例1の結果を図5に示す。なお、図4及び図5中、横軸(log 2 expression of control experiment)は、コントロール検体における発現量(log2)を表し、縦軸(log 2 fold change)は、コントロールに対しての発現変化の割合(log2)を表す。
3’-メチル-ヌクレオチドである、(2R,3S,5R)-2-(4,4’-ジメトキシトリチルオキシメチル)-3-メチル-5-(5-メチル-2,4-ジオキソ-3,4-ジヒドロピリミジン-1(2H)-イル)テトラヒドロフラン-3-イル(2-シアノエチル)ホスホルアミダイトは、Journal of the Chemical Society, Perkin Transactions 1,1998, 120, pp 5458-5463に記載の方法に準じて合成した。
表10に記載されたアンチセンスオリゴヌクレオチドを、核酸自動合成機を使用して調製した。標的遺伝子は、実施例2および比較例2と同様に、マウスcoagulation factor XI(FXI)である。
合成したオリゴヌクレオチドの分子量は、MALDI-TOF-MASSにより測定した。結果を表10に示す。
評価例5と同じ評価方法を用いて、RNAの切断活性を測定した。反応時間は1.5時間とした。
Claims (31)
- 中央領域、5’側領域及び3’側領域を有するアンチセンスオリゴヌクレオチドであって、
ここで、
- 中央領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群より独立して選択される少なくとも5個のヌクレオチドからなり、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から選択される糖部修飾ヌクレオチドを少なくとも一つ含み、その3’末端及び5’末端が、それぞれ独立して、デオキシリボヌクレオチド、リボヌクレオチド、2’-3’架橋ヌクレオチド又は3’位修飾非架橋ヌクレオチドであり、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を、少なくとも一つ含み;
- 5’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その3’末端が、糖部修飾ヌクレオチドであり、ここで、該3’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まず;
- 3’側領域は、
デオキシリボヌクレオチド、リボヌクレオチド及び糖部修飾ヌクレオチドからなる群から独立して選択される少なくとも1個のヌクレオチドからなり、その5’末端が、糖部修飾ヌクレオチドであり、ここで、該5’末端の糖部修飾ヌクレオチドは、中央領域に結合し、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドを除く糖部修飾ヌクレオチドから選択され、そして
デオキシリボヌクレオチド、2’-3’架橋ヌクレオチド及び3’位修飾非架橋ヌクレオチドからなる群から独立して選択される、連続する少なくとも4個のヌクレオチドから構成されるオリゴヌクレオチド鎖を含まない、
アンチセンスオリゴヌクレオチド。 - 前記中央領域が、5~15個のヌクレオチドからなり、
前記5’側領域及び前記3’側領域が、それぞれ独立して、1~7個のヌクレオチドからなる、請求項1に記載のアンチセンスオリゴヌクレオチド。 - 前記中央領域が、8~12個のヌクレオチドからなり、
前記5’側領域及び前記3’側領域が、それぞれ独立して、2~5個のヌクレオチドからなる、請求項1又は2に記載のアンチセンスオリゴヌクレオチド。 - 中央領域に含まれる、2’-3’架橋ヌクレオチドが、
下記式(I):
(式中、mは、1、2、3又は4であり、
Bxは、核酸塩基部分であり、
Xは、O又はSであり、
-Q-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であり、
mが、2、3又は4であるとき、2つの隣り合った-Q-は、一緒に
式 -CR7=CR8-
で表される基を形成してもよく、
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;
R6は、C1-C12アルキル又はアミノ保護基であり、
R7及びR8は、それぞれ独立して、水素原子又はC1-C6アルキルである)
で表される部分構造を含むヌクレオチドである、請求項1から3の何れか一つに記載のアンチセンスオリゴヌクレオチド。 - 中央領域に含まれる、3’位修飾非架橋ヌクレオチドが、
下記式(II):
(式中、Bxは、核酸塩基部分であり、
Xは、O又はSであり、
R12は、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
R1、R2、R3及びR11は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され;
J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基である)
で表される部分構造を含むヌクレオチドである、請求項1から3の何れか一つに記載のアンチセンスオリゴヌクレオチド。 - 中央領域に含まれる、2’-3’架橋ヌクレオチドが、
下記式(III):
(式中、Bxは、核酸塩基部分であり、
Xは、O又はSであり、
-Q1-及び-Q2-は、それぞれ独立して、-CR4R5-、-C(=O)-、-C(=S)-、-C(=NR6)-、-O-、-NH-、-NR6-又は-S-であるか、あるいは、
-Q1-Q2-は、-CR7=CR8-であり;かつ、式中、R7及びR8は独立して水素原子又はC1-C6アルキルであり、
R1、R2、R3、R4及びR5は、それぞれ独立して、水素原子、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニル、1つ以上の置換基で置換されたC1-C6アルキル、1つ以上の置換基で置換されたC2-C6アルケニル、1つ以上の置換基で置換されたC2-C6アルキニル、アシル、1つ以上の置換基で置換されたアシル、1つ以上の置換基で置換されたアミド、ヒドロキシ、C1-C6アルコキシ、1つ以上の置換基で置換されたC1-C6アルコキシ、スルファニル、C1-C6アルキルチオ又は1つ以上の置換基で置換されたC1-C6アルキルチオであり;ここで、前記置換基は、それぞれ独立して、ハロゲン原子、オキソ、OJ1、NJ1J2、SJ1、アジド、OC(=Y)J1、OC(=Y)NJ1J2、NJ3C(=Y)NJ1J2及びシアノからなる群から独立して選択され、J1、J2及びJ3は、それぞれ独立して、水素原子又はC1-C6アルキルであり、かつYは、O、S又はNJ4であり、J4はC1-C12アルキル又はアミノ保護基であり;
R6は、C1-C12アルキル又はアミノ保護基である)
で表されるヌクレオチドである、請求項4に記載のアンチセンスオリゴヌクレオチド。 - -Q1-が、-O-、-NH-、-NR6-又は-S-であり、R6はC1-C12アルキルであり、-Q2-が-CH2-である、請求項6に記載のアンチセンスオリゴヌクレオチド。
- -Q1-が、-O-であり、-Q2-が-CH2-である、請求項6又は7に記載のアンチセンスオリゴヌクレオチド。
- R1、R2及びR3が、水素原子である、請求項4から8の何れか一つに記載のアンチセンスオリゴヌクレオチド。
- Xが、Oである、請求項4から9の何れか一つに記載のアンチセンスオリゴヌクレオチド。
- 前記中央領域が、ギャップ領域であり、
前記5’側領域が、5’ウィング領域であり、
前記3’側領域が、3’ウィング領域である、請求項1から10の何れか一つに記載のアンチセンスオリゴヌクレオチド。 - 前記5’側領域及び前記3’側領域に含まれる糖部修飾ヌクレオチドが、それぞれ独立して、2’位修飾非架橋ヌクレオチド及び2’,4’-BNAからなる群から選択される、請求項1から11の何れか一つに記載のアンチセンスオリゴヌクレオチド。
- 前記2’位修飾非架橋ヌクレオチドが、2’-O-メチル化ヌクレオチド、2’-O-メトキシエチル(MOE)化ヌクレオチド、2’-O-アミノプロピル(AP)化ヌクレオチド、2’-フルオロ化ヌクレオチド、2’-O-(N-メチルアセトアミド)(NMA)化ヌクレオチド及び2’-O-メチルカルバモイルエチル(MCE)化ヌクレオチドからなる群から選択される少なくとも1つである、請求項12に記載のアンチセンスオリゴヌクレオチド。
- 前記2’,4’-BNAが、LNA、cEt-BNA、ENA、BNANC、AmNA及びscpBNAからなる群から選択される少なくとも1つである、請求項12に記載のアンチセンスオリゴヌクレオチド。
- アンチセンスオリゴヌクレオチドが、ホスホロチオエート結合を含む、請求項1から14の何れか一つに記載のアンチセンスオリゴヌクレオチド。
- 標識機能、精製機能及び標的部位への送達機能からなる群から選択される少なくとも1種の機能を有する機能性分子に由来する基をさらに含む、請求項1から15の何れか一つに記載のアンチセンスオリゴヌクレオチド。
- 前記機能性分子が、糖、脂質、ペプチド及びタンパク質並びにそれらの誘導体からなる群から選択される、請求項16に記載のアンチセンスオリゴヌクレオチド。
- 前記機能性分子が、コレステロール、トコフェロール及びトコトリエノールからなる群から選択される脂質である、請求項16又は17に記載のアンチセンスオリゴヌクレオチド。
- 前記機能性分子が、アシアロ糖タンパク質受容体と相互作用する糖誘導体である、請求項16又は17に記載のアンチセンスオリゴヌクレオチド。
- 前記機能性分子が、受容体のリガンド及び抗体からなる群から選択される、ペプチド又はタンパク質である、請求項16又は17に記載のアンチセンスオリゴヌクレオチド。
- 請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチドのプロドラッグ。
- (i)請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチド及び
(ii)少なくとも1個のリボヌクレオチドを含み、かつ前記(i)アンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチド
を含む、オリゴヌクレオチド複合体。 - (i)請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチドに由来する基、及び
(ii)少なくとも1個のリボヌクレオチドを含み、かつ前記(i)のアンチセンスオリゴヌクレオチドに対してハイブリダイズする領域を含むオリゴヌクレオチドに由来する基
を含み、前記(i)アンチセンスオリゴヌクレオチドに由来する基と、前記(ii)オリゴヌクレオチドに由来する基が、連結されたオリゴヌクレオチド。 - (iii) 請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチドに由来する基に、少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖が連結されたオリゴヌクレオチド、及び
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチド
を含むオリゴヌクレオチド複合体であって、
前記(iii)の少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含む前記オリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド複合体。 - (iii) 請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチドに由来する基に、少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖が連結されたオリゴヌクレオチドに由来する基、及び
(iv)RNaseHによって認識される少なくとも4個の連続するヌクレオチドを含むオリゴヌクレオチド鎖を含むオリゴヌクレオチドに由来する基
を含み、前記(iii)オリゴヌクレオチドに由来する基と、前記(iv)オリゴヌクレオチドに由来する基が、連結され、
前記(iii)の少なくとも1個のリボヌクレオチドを含むオリゴヌクレオチド鎖と、前記(iv)のRNaseHによって認識される少なくとも4個の連続するヌクレオチドを含む前記オリゴヌクレオチド鎖とがハイブリダイズする、オリゴヌクレオチド。 - 請求項1から25の何れか一つに記載のアンチセンスオリゴヌクレオチド、請求項21に記載のプロドラッグ、請求項22若しくは24に記載のオリゴヌクレオチド複合体、又は請求項23若しくは25に記載のオリゴヌクレオチドと、薬理学上許容される担体とを含む、医薬組成物。
- 請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチド、請求項21に記載のプロドラッグ、請求項22若しくは24に記載のオリゴヌクレオチド複合体、又は請求項23若しくは25に記載のオリゴヌクレオチドと、細胞とを接触させる工程を含む、標的RNAの機能を制御する方法。
- 請求項26に記載の医薬組成物を、哺乳動物に投与する工程を含む、該哺乳動物における標的RNAの機能を制御する方法。
- 請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチド、請求項21に記載のプロドラッグ、請求項22若しくは24に記載のオリゴヌクレオチド複合体、又は請求項23若しくは25に記載のオリゴヌクレオチドと、細胞とを接触させる工程を含む、標的遺伝子の発現を制御する方法。
- 請求項26に記載の医薬組成物を、哺乳動物に投与する工程を含む、該哺乳動物における標的遺伝子の発現を制御する方法。
- 2’-3’架橋ヌクレオチド及び、3’位修飾非架橋ヌクレオチドからなる群から選択されるヌクレオチドを使用して、請求項1から20の何れか一つに記載のアンチセンスオリゴヌクレオチド又は請求項21に記載のプロドラッグを製造する方法。
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WO2023112931A1 (ja) * | 2021-12-13 | 2023-06-22 | 日本新薬株式会社 | ATN1 mRNA又はpre-mRNAを標的とするアンチセンスオリゴヌクレオチド |
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