Antisense oligonucleotide

Abstract

The present invention relates to an antisense oligonucleotide having one or more phosphorothioate bonds. An aTNA structure represented by general formula (A1) or (A2) [in the general formula, R0 is a C1-30 alkyl group that is substituted with one or more fluorine atoms and may have an ether-bondable oxygen atom, or is a C10-30 alkyl group that is not substituted with a fluorine atom and may have an ether-binding oxygen atom; n11 and n12 are each independently an integer of 1 or greater; B is a nucleobase; and a black dot represents a bonding site] is provided on least one terminal end of the antisense oligonucleotide.

Description

Antisense oligonucleotides

[0001] The present invention relates to an antisense oligonucleotide (hereinafter sometimes abbreviated as "ASO") used for RNA interference, and a pharmaceutical composition containing said antisense oligonucleotide as an active ingredient. This application claims priority based on Japanese Patent Application No. 2025-050754, filed in Japan on March 25, 2025, the contents of which are incorporated herein by reference.

[0002] In recent years, research into nucleic acid drugs using oligonucleotides has been progressing. Nucleic acid drugs have advantages such as high specificity for target molecules and fewer side effects. However, nucleic acid drugs have low cell membrane permeability, making it difficult to deliver them to target molecules present inside cells. Drug delivery agents using lipid nanoparticles (Patent Document 1) and cationic polymer nanoparticles (Patent Document 2) are known. However, there is much room for improvement in terms of cell membrane permeability efficiency and toxicity concerns.

[0003] Furthermore, because nucleic acids have phosphodiester bonds that are easily degraded by nucleases, nucleic acid drugs also face the challenge of stability when administered to the body. If stability is low, the drug may be degraded in the body before reaching the target tissue, and the desired therapeutic effect cannot be obtained. Generally, many ASOs and siRNAs used in nucleic acid drugs undergo various modifications to enhance their stability in the body. Among the modifications used in nucleic acid drugs, modifications to the 2' position of deoxyribose (ribose in the case of RNA) in the nucleotide include modifications that introduce a methoxyethyloxy group to the 2' position (2'-MOE modification), modifications that introduce a methoxy group to the 2' position (2'-OMe modification), and modifications that introduce a fluorine atom to the 2' position (2'-F modification). Modifications to the phosphodiester bond include modifications that replace one of the oxygen atoms in the phosphodiester bond with a methyl group to form a methylphosphonate bond, modifications that introduce an alkyl group to one of the oxygen atoms to form a phosphotriester bond, and modifications that replace one of the oxygen atoms with a sulfur atom to form a phosphorothioate bond (PS modification). In addition, artificial nucleic acids that exhibit superior nuclease resistance and other properties compared to natural nucleic acids are also used. Examples of such artificial nucleic acids include acyclic glycol nucleic acids (GNA), peptide nucleic acids (PNA), acyclic threoninol nucleic acids (aTNA), and serinol nucleic acids (SNA) (Non-Patent Literature 1).

[0004] On the other hand, compounds having a polyfluoro structure are known to be stable and low-toxicity in vivo, and to be excellent in terms of intracellular uptake and exit from endosomes (Non-Patent Document 1). Taking advantage of this property, it has been reported that the cell membrane permeability of acyclic threoninol-type nucleic acids (aTNA-type nucleic acids) can be improved by introducing a fluoroalkyl group into them (Patent Document 3), and that by linking the nucleic acid to be introduced into cells with the aTNA-type nucleic acid to which the fluoroalkyl group has been introduced, the cell membrane permeability of the nucleic acid is improved, making it possible to introduce it into cells without the use of transfection reagents (Patent Document 4).

[0005] International Publication No. 2011 / 036557, International Publication No. 2017 / 212006, International Publication No. 2022 / 186350, International Publication No. 2024 / 038781

[0006] Murayama et al., Chemistry A European Journal, 2013, vol.19, p.14151-14158.Zhang et al., MRS Communications, 2018, vol.8, p.303-313.

[0007] The present invention aims to provide an ASO that exhibits excellent intracellular introduction efficiency and knockdown efficiency, and a pharmaceutical composition containing the ASO.

[0008] The inventors of the present invention have discovered that by attaching an aTNA-type nucleic acid with a fluoroalkyl group introduced to the terminal end of a PS-modified ASO, both the efficiency of introduction into cells and the knockdown efficiency are improved, thus completing the present invention.

[0009] In other words, the present invention includes the following embodiments: [1] An antisense oligonucleotide having one or more phosphorothioate bonds, wherein one of the ends of the antisense oligonucleotide is a product of the following general formula (A1) or (A2)

[0010]

[0011] [In the formula, R ０An antisense oligonucleotide having an aTNA structure represented by [1], wherein n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; and black circles indicate bonding hands. [2] The antisense oligonucleotide of [1], wherein at least one of the bonds between the 1 to 5 nucleotides at the terminal end of the antisense oligonucleotide that is not to which the aTNA structure is bonded is a phosphorothioate bond. [3] The antisense oligonucleotide of [1] or [2], wherein the antisense oligonucleotide has one or more phosphodiester bonds. [4] An antisense oligonucleotide from any of [1] to [3], wherein all bonds between the nucleotides constituting the antisense oligonucleotide are phosphorothioate bonds. [5] An antisense oligonucleotide from any of [1] to [4], wherein the 2' position of the deoxyribose or ribose in all nucleosides constituting the antisense oligonucleotide is unmodified. [6] An antisense oligonucleotide from any of [1] to [5], wherein n11 or n12 is 2 or more. [7] An antisense oligonucleotide from any of [1] to [6], wherein n11 or n12 is 2 or 3. [8] The R ０ However, any of the antisense oligonucleotides [1] to [7] above, which are alkyl groups having 1 to 30 carbon atoms substituted with at least two fluorine atoms. [9] The R ０ However, any of the antisense oligonucleotides from [1] to [7] above, which is a perfluoroalkyl group having 1 to 10 carbon atoms, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a perfluoroalkyl group having 2 to 10 carbon atoms.

[10] The R０ An antisense oligonucleotide from any of [1] to [7], wherein the antisense oligonucleotide is an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom.

[11] A pharmaceutical composition comprising an antisense oligonucleotide from any of [1] to

[10] , a pharmacoacceptable salt thereof, or a hydrate thereof.

[12] The pharmaceutical composition from

[11] used in gene therapy.

[13] Use of an antisense oligonucleotide from any of [1] to

[10] , a pharmacoacceptable salt thereof, or a hydrate thereof for the manufacture of a gene therapy agent.

[0012] The present invention provides an ASO that exhibits excellent intracellular introduction efficiency and knockdown efficiency, as well as a pharmaceutical composition containing the ASO.

[0013] In the present invention and this specification, "nucleic acid" means a molecule in which nucleotides are linked via phosphodiester bonds. This includes not only naturally occurring nucleotides such as DNA and RNA, but also artificial nucleotides that are modified from naturally occurring nucleotides and capable of being linked to them via phosphodiester bonds. Examples of artificial nucleotides include those in which the side chains of naturally occurring nucleotides are modified with functional groups such as amino groups, those in which the hydroxyl group at the 2' position of the ribose skeleton is replaced with a methoxy group, fluoro group, methoxyethyl group, etc., phosphorothioate nucleotides (in which the oxygen atom of the phosphate group is replaced with a sulfur atom), morpholino nucleotides (in which ribose or deoxyribose is replaced with a morpholin ring), BNA (Bridged nucleic acid), HNA (Hexitol Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), TNA (Threose nucleic acid), GNA (Glycerol nucleic acid), and CeNA (Cyclohexenyl nucleic acid). Furthermore, "nucleic acids" include molecules in which only one or more natural nucleotides are linked by phosphodiester bonds, such as DNA and RNA; molecules in which one or more natural nucleotides and one or more artificial nucleotides are linked via phosphodiester bonds; and molecules in which only one or more artificial nucleotides are linked by phosphodiester bonds. In addition, molecules in which one or more natural nucleosides and / or artificial nucleosides are selected from the group consisting of phosphorothioate bonds, methylphosphonate bonds, and phosphotriester bonds are also included in "nucleic acids."

[0014] In the present invention and the specification of the present application, the term "antisense nucleotide" means a single-stranded nucleic acid having a nucleotide sequence that is sufficiently complementary to be capable of associating with a target region in a target mRNA. The target region in the target mRNA is referred to as a "sense region", and the region in the antisense nucleotide that associates with the target region in the target mRNA is referred to as an "antisense region". In many antisense nucleotides, the nucleotide sequence of the antisense region is a sequence complementary to the nucleotide sequence of the sense region, but it may also contain mismatched bases.

[0015] In the present invention and the specification of the present application, "C ｐ１－ｐ２ " (where p1 and p2 are positive integers satisfying p1 < p2) means a group having a carbon number of p1 to p2.

[0016] In the present invention and the specification of the present application, "C １－３０ alkyl group" means an alkyl group having 1 to 30 carbon atoms, which may be linear or branched. "C ２－３０ alkyl group" means an alkyl group having 2 to 30 carbon atoms, which may be linear or branched. Examples of C １－３０ alkyl groups include methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, heptyl group, octyl group, nonyl group, decyl group, undecyl group, dodecyl group, tridecyl group, tetradecyl group, pentadecyl group, hexadecyl group, heptadecyl group, octadecyl group, nonadecyl group, eicosyl group, heneicosyl group, docosyl group, tricosyl group, tetracosyl group, pentacosyl group, hexacosyl group, heptacosyl group, octacosyl group, nonacosyl group, triacontyl group, and the like.

[0017] In the present invention and the specification of the present application, "C １－２０ alkyl group" means an alkyl group having 1 to 20 carbon atoms, which may be linear or branched. "C ２－２０ alkyl group" means an alkyl group having 2 to 20 carbon atoms, which may be linear or branched. C １－２０Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, and eicosyl groups.

[0018] In the present invention and this specification, "C １－１０ "Alkyl alkyl group" is an alkyl group having 1 to 10 carbon atoms, and may be a straight chain or a branched chain. ２－１０ An alkyl group is an alkyl group having 2 to 10 carbon atoms, and may be a straight chain or a branched chain. １－１０ Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, and decyl groups.

[0019] In the present invention and this specification, "C １０－３０ An alkyl group is an alkyl group having 10 to 30 carbon atoms, and may be a straight chain or a branched chain. １０－３０ Examples of alkyl groups include undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0020] In the present invention and this specification, "C １－６ An alkyl group is an alkyl group having 1 to 6 carbon atoms, and may be a straight chain or a branched chain. １－６Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl groups.

[0021] In the present invention and this specification, "alkylene group" is a divalent group obtained by removing two hydrogen atoms from a saturated hydrocarbon, and may be a linear or branched chain. Examples of alkylene groups include methylene group, ethylene group, trimethylene group, tetramethylene group, pentamethylene group, hexamethylene group, heptamethylene group, octamethylene group, nonamethylene group, methylmethylene group, ethylmethylene group, methylethylene group, methylpropylene group, ethylethylene group, dimethylmethylene group, 1,2-dimethylethylene group, 1,1-dimethylethylene group, 1-ethylpropylene group, 2-ethylpropylene group, 1,2-dimethylpropylene Examples include the 1-propylpropylene group, 2,2-dimethylpropylene group, 1-propylpropylene group, 2-propylpropylene group, 1-methyl-1-ethylpropylene group, 1-methyl-2-ethylpropylene group, 1-ethyl-2-methylpropylene group, 2-methyl-2-ethylpropylene group, 1-methylbutylene group, 2-methylbutylene group, 3-methylbutylene group, 2-ethylbutylene group, 1-methylpentylene group, 2-ethylpentylene group, and 1-methylhexylene group.

[0022] In the present invention and this specification, "C １－２０ A "perfluoroalkyl group" is a group in which all hydrogen atoms of an alkyl group having 1 to 20 carbon atoms are replaced with fluorine atoms. １－１０Examples of perfluoroalkyl groups include perfluoromethyl, perfluoroethyl, perfluoropropyl, perfluoroisopropyl, perfluorobutyl, perfluoroisobutyl, perfluorosec-butyl, perfluorotert-butyl, perfluoropentyl, perfluoroisopentyl, perfluoroneopentyl, perfluorotert-pentyl, perfluorohexyl, perfluoroheptyl, perfluorooctyl, perfluorononyl, perfluorodecyl, perfluoroundecyl, perfluorododecyl, perfluorotridecyl, perfluorotetradecyl, perfluoropentadecyl, perfluorohexadecyl, perfluoroheptadecyl, perfluorooctadecyl, perfluorononadecyl, and perfluoroeicosyl groups.

[0023] In the present invention and this specification, a "perfluoroalkylene group" is a group in which all hydrogen atoms of an alkylene group are replaced with fluorine atoms. An example of a perfluoroalkylene group is the group in which all hydrogen atoms of the alkylene group mentioned above are replaced with fluorine atoms.

[0024] In the present invention and this specification, "ether-bonded oxygen atom" refers to an oxygen atom that links carbon atoms together, and does not include oxygen atoms linked in series. The maximum number of ether-bonded oxygen atoms that an alkyl group with Nc (where Nc is an integer of 2 or more) may have is Nc-1. Furthermore, "a C atom having an ether-bonded oxygen atom between carbon atoms" ２－１０ "Alkyl alkyl group" refers to C ２－１０ This group has at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group. Hereinafter, alkyl groups having an ether-bonded oxygen atom may be referred to as "ether-bonded alkyl groups."

[0025] In the present invention and this specification, "having an ether-bonded oxygen atom between carbon atoms, C ２－１０ "Perfluoroalkyl group" is C ２－１０ Ether bond-containing C having at least one ether-bonded oxygen atom between the carbon atoms of the alkyl group２－１０ This is a group in which all hydrogen atoms of the alkyl group are replaced with fluorine atoms. Hereinafter, perfluoroalkyl groups having ether-bonded oxygen atoms may be referred to as "ether-bonded perfluoroalkyl groups."

[0026] In the present invention and this specification, "halogen atom" means a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom. "Halogen atom other than a fluorine atom" means a chlorine atom, a bromine atom, or an iodine atom. Examples of "halogen atoms other than a fluorine atom" are preferably chlorine atoms or bromine atoms, and chlorine atoms are particularly preferred.

[0027] Furthermore, in the following, "compound (X)" refers to the compound represented by formula (X).

[0028] <Antisense Oligonucleotide (ASO)> The ASO according to this embodiment is an ASO having one or more phosphorothioate bonds, and having an aTNA structure represented by the following general formula (A1) or (A2) at one of the ends of the ASO.

[0029]

[0030] [In the formula, R ０ [The group is an alkyl group having 1 to 30 carbon atoms substituted with one or more fluorine atoms, a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 2 to 30 carbon atoms substituted with one or more fluorine atoms, an alkyl group having 10 to 30 carbon atoms not substituted with fluorine atoms, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms not substituted with fluorine atoms; n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; black circles indicate bonds]

[0031] Hereafter, the aTNA structure represented by general formula (A1) may be referred to as "aTNA structure (A1)," and the aTNA structure represented by general formula (A2) may be referred to as "aTNA structure (A2)." Furthermore, hereafter, both aTNA structure (A1) and aTNA structure (A2) will be collectively referred to as "RF-aTNA structure."

[0032] In general formulas (A1) and (A2), R ０ C is a carbon atom substituted with at least one fluorine atom. １－３０ Alkyl(C) １－３０ (Fluoroalkyl groups), or C atoms not substituted with a fluorine atom １０－３０ It is an alkyl group. If the alkyl group has two or more carbon atoms, it may have one to five ether-bonded oxygen atoms between carbon atoms. In the present invention and this specification, "C １－３０ Fluoroalkyl groups (if the alkyl group has two or more carbon atoms, the alkyl group may have one to five ether-bonded oxygen atoms between carbon atoms) are defined as "C １－３０ Fluoroalkyl groups, or C ２－３０ This refers to a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of a fluoroalkyl group.

[0033] R ０ C １－３０ In the case of fluoroalkyl groups, one or more hydrogen atoms bonded to the carbon atom may be further substituted with halogen atoms other than fluorine atoms. Structure (A1) or structure (A2) is R ０ C is a carbon atom substituted with at least two fluorine atoms. １－３０ It is preferable that it be a fluoroalkyl group. In particular, R ０ C １－２０ Fluoroalkyl groups are preferred, C １－１５ Fluoroalkyl groups are more preferred, C ２－１５ A fluoroalkyl group is more preferably C ６－10 Fluoroalkyl groups are even more preferred. ０ C １－３０ In the case of fluoroalkyl groups, the number of hydrogen atoms substituted for fluorine atoms is not particularly limited as long as there is one or more; for example, three or more are preferred, six or more are more preferred, and seven or more are even more preferred.

[0034] R ０ C １－３０ In the case of a fluoroalkyl group, R ０Specifically, a group represented by the following general formula (f-1) or (f-2) is preferred. Here, Rf Ｐ is a completely halogenated C １－２０ Alkyl(C) １－２０ A group in which all of the hydrogen atoms of an alkyl group are replaced by halogen atoms, and which has one or more fluorine atoms. Rf Ｐ If the carbon number is 2 or more, that is, completely halogenated C ２－２０ In the case of alkyl groups, there may be 1 to 5 ether-bonded oxygen atoms between the carbon atoms. In the present invention and this specification, "a fully halogenated C which may have 1 to 5 ether-bonded oxygen atoms between the carbon atoms" ２－２０ "Alkyl alkyl group" refers to "completely halogenated C ２－２０ Alkyl alkyl groups, or fully halogenated C ２－２０ This refers to a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group.

[0035] In the general formula (f-2), two Rf Ｐ These groups may be of the same kind or different kinds. Rf Ｐ C １－２０ Perfluoroalkyl groups (C １－２０ It is preferable that the alkyl group is a group in which all of the hydrogen atoms are replaced by fluorine atoms.

[0036] In the following general formulas (f-1) or (f-2), n1 is an integer from 0 to 10, and n2 is an integer from 0 to 9. When both n1 and n2 are 0, both represent a simple combination. That is, when n1 is 0, the base represented by general formula (f-1) is -Rf Ｐ And when n2 is 0, the group represented by the general formula (f-2) is -CH(Rf Ｐ ) ２ That is the case.

[0037]

[0038] R ０ If the group is represented by the general formula (f-1), then R ０ Rf ＰThe group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 4. Ｐ However, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n1 is an integer from 0 to 2. Ｐ However, the group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n1 is an integer from 0 to 2 (however, when n1 is 1, Rf Ｐ More preferably, Rf (excluding groups where is a trifluoromethyl group) Ｐ However, a group that is a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, a perfluorohexyl group, a perfluoroheptyl group, a perfluorooctyl group, a perfluorononyl group, or a perfluorodecyl group, where n1 is 0, is even more preferred.

[0039] R ０ If the group is represented by the general formula (f-2), then R ０ Rf Ｐ The group is preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 4. ＰHowever, the group is more preferably a trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, or perfluorodecyl group, where n2 is an integer from 0 to 2. Ｐ The group is a trifluoromethyl group, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is an integer from 0 to 2 (however, n2 is 0 or 1, and Rf Ｐ More preferably, Rf (excluding groups where is a trifluoromethyl group) Ｐ However, a pentafluoroethyl group, a heptafluoropropyl group, a nonafluorobutyl group, a perfluoropentyl group, or a perfluorohexyl group, where n2 is 0, is even more preferred.

[0040] R ０ C １－３０ In the case of a fluoroalkyl group, R ０ Examples include trifluoromethyl group, pentafluoroethyl group, heptafluoropropyl group, nonafluorobutyl group, perfluoropentyl group, perfluorohexyl group, perfluoroheptyl group, perfluorooctyl group, perfluorononyl group, perfluorodecyl group, difluoromethyl group, 1,1-difluoroethyl group, 2,2-difluoroethyl group, 1,1,2,2-tetrafluoroethyl group, 1, Examples of the RF-aTNA structure in the ASO according to this embodiment include 1,2,2,3,3-hexafluoropropyl group, 1,1,2,3,3,3-hexafluoropropyl group, 1,1,2,2,3,3-hexafluorohexyl group, 1,1,2,2,3,3-hexafluorooctyl group, 1,1,2,2,3,3-hexafluorodecyl group, 1,1,2,2,3,3-hexafluorooctadecyl group, 1,1,2,2,3,3-hexafluorohexacosyl group, etc. In the ASO according to this embodiment, R is the R in the general formula (A1) or (A2). ０ C １－３０ A perfluoroalkyl group structure is preferred, and in general formula (A1) or (A2), R ０ C １－２０A structure that is a perfluoroalkyl group is more preferable, and in general formula (A1) or (A2), R ０ is a C １－１０ perfluoroalkyl group, and a structure that is a perfluoroalkyl group is even more preferable.

[0041] R ０ is a C １０－３０ alkyl group, as structure (A1) or structure (A2), R ０ is C １０－２５ alkyl group is preferable, C １５－２５ alkyl group is more preferable, and C １５－２３ alkyl group is even more preferable. An alkyl group of sufficient length has high hydrophobicity similarly to a fluoroalkyl group, and contributes to the cell membrane permeability of a nucleic acid having structure (A1) or structure (A2).

[0042] In general formula (A2), B is a nucleic acid base. Preferred nucleic acid bases include those found in natural nucleic acids, nucleic acid bases structurally similar to natural nucleic acid bases, and modified bases that have undergone various modifications. Modifications of the base include alkylation, hydroxylation, alkoxylation, acylation, dihydrolation, amination, formylation, halogenation, etc. Nucleic acid bases structurally similar to natural nucleic acid bases include triazole, imidazole, azapyrimidine, azapurine, etc. Specifically, the nucleic acid base of B is adenine, guanine, cytosine, thymine, uracil, 1-methyladenine, N6-methyladenine, 7-methylguanine, 5-methylcytosine, 1-methylthymine, 5-methyluracil, 5-hydroxymethylcytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, dihydrouracil, dihydrothymine, dihydrocytosine, 2,6-diaminoadenine, 2,6-diamino Examples include guanine, 6-thioguanine, 2-thioadenine, 2-thiocytosine, 4-thiouracil, 5-fluorouracil, 5-iodouracil, 5-halogenocytosine, 5-fluorocytosine, 5-trihalogenomethyluracil, 5-trifluoromethyluracil, 5-azathymine, 5-azacytosine, 6-azauracil, 8-azaadenine, 7-deazaadenine, 7-deazaguanine, and 3-deazauracil. The aTNA structure (A2) in the ASO according to this embodiment is preferably a nucleic acid having a structure in which B in general formula (A2) is adenine, guanine, cytosine, thymine, or uracil.

[0043] In general formulas (A1) and (A2), n11 and n12 are each independent integers of 1 or more. n11 and n12 are the number of repeats per molecule of each structure, and the larger the number, the higher the hydrophobicity. In the ASO according to this embodiment, it is preferable that n11 and n12 are 2 or more. Furthermore, since it is easier to obtain structures similar to natural double-stranded nucleic acids and single-stranded nucleic acids, it is preferable that n11 and n12 are 10 or less, more preferably 8 or less, and even more preferably 5 or less. In the ASO according to this embodiment, it is preferable that n11 is 2 or more and 10 or less, more preferably 2 or more and 8 or less, even more preferably 2 or more and 5 or less, and particularly preferable 2 or 3. When n11 and n12 are 2 or more, the multiple structures (A1) or structures (A2) may be the same as each other or may be different structures.

[0044] In this embodiment, one or more bonds between the nucleotides constituting the ASO are phosphorothioate bonds. In addition to the RF-aTNA structure, the ASO in this embodiment achieves high knockdown efficiency because at least a portion of the nucleotide binding portion (the portion corresponding to the phosphodiester bond portion in the natural nucleotide) is PS modified.

[0045] The number of phosphorothioate bonds in the ASO according to this embodiment may be at least one, or may be three to ten, and all nucleotide binding sites in the ASO may be phosphorothioate bonds. In the ASO according to this embodiment, when the ratio of the number of phosphorothioate bonds to the total number of nucleotide binding sites in the ASO is defined as the PS modification rate (%) ([PS modification rate (%)] = [Number of phosphorothioate bonds in the ASO] / [Total number of nucleotide binding sites in the ASO] × 100%), the PS modification rate (%) of the ASO according to this embodiment is preferably 10 to 100%, more preferably 20 to 100%, even more preferably 30 to 100%, and even more preferably 50 to 100%.

[0046] Generally, degradation by nucleases occurs from the terminal portion of the oligonucleotide. Therefore, in the case of the ASO according to this embodiment, where a portion of all nucleotide binding sites in the ASO is a phosphorothioate bond and there is one or more phosphodiester bonds, it is preferable that at least one nucleotide binding site on the terminal side of the ASO that is not bound to the RF-aTNA structure is a phosphorothioate bond, more preferably that at least one of the 1 to 5 nucleotide binding sites on the terminal side that is not bound to the RF-aTNA structure is a phosphorothioate bond, and even more preferably that two or more of the 1 to 5 nucleotide binding sites on the terminal side that is not bound to the RF-aTNA structure are phosphorothioate bonds. In this embodiment, the ASO is preferably such that the first nucleotide bond from the end not to which the RF-aTNA structure is attached is a phosphorothioate bond, more preferably such that the first nucleotide bond from the end not to which the RF-aTNA structure is attached and at least one of the 2nd to 5th nucleotide binding portions from that end are phosphorothioate bonds, even more preferably such that the first nucleotide bond from the end not to which the RF-aTNA structure is attached and two or more of the 2nd to 5th nucleotide binding portions from that end are phosphorothioate bonds, and even more preferably such that all 1 to 5 nucleotide binding portions at the end not to which the RF-aTNA structure is attached are phosphorothioate bonds.

[0047] In this embodiment, if a portion of all nucleotide binding sites in the ASO is a phosphorothioate bond and the ASO has one or more phosphodiester bonds, then at least one nucleotide binding site at the end of the ASO where the RF-aTNA structure is attached may be a phosphorothioate bond, at least one of the 1 to 5 nucleotide binding sites at the end where the RF-aTNA structure is attached may be a phosphorothioate bond, it is more preferable that the first nucleotide binding site from the end where the RF-aTNA structure is attached is a phosphorothioate bond, and it is even more preferable that all 1 to 5 nucleotide binding sites at the end where the RF-aTNA structure is attached are phosphorothioate bonds.

[0048] In this embodiment, if a portion of all nucleotide binding sites in the ASO is a phosphorothioate bond and the ASO has one or more phosphodiester bonds, then at least one nucleotide binding site at the end of the ASO where the RF-aTNA structure is attached may be a phosphodiester bond, at least one of the 1 to 5 nucleotide binding sites at the end where the RF-aTNA structure is attached may be a phosphodiester bond, and two or more of the 1 to 5 nucleotide binding sites at the end where the RF-aTNA structure is attached may be phosphodiester bonds.

[0049] In the present embodiment, if a portion of all nucleotide binding portions of the ASO is a phosphorothioate bond and the ASO has one or more phosphodiester bonds, it is preferable that the phosphodiester bonds are located near the center of the ASO; more preferably that the nucleotide binding portions in both terminal regions of the ASO are phosphorothioate bonds and the nucleotide binding portions in the central region of the ASO are phosphodiester bonds; even more preferably that at least one of the 1 to 5 nucleotide binding portions at both ends of the ASO is a phosphorothioate bond and the remaining nucleotide binding portions are phosphodiester bonds; and even more preferably that the 1 to 5 nucleotide binding portions at both ends of the ASO are phosphorothioate bonds and the remaining nucleotide binding portions are phosphodiester bonds.

[0050] In this embodiment, it is preferable that the 2' position (hereinafter sometimes abbreviated as "2' position") of the deoxyribose or ribose in all nucleotides constituting the ASO is unmodified, and it is more preferable that all nucleosides constituting the ASO are natural-type nucleosides. In this embodiment, sufficient knockdown efficiency can be achieved even if the 2' position is unmodified by introducing the RF-aTNA structure and PS modification. Furthermore, in this embodiment, if all nucleosides constituting the ASO are natural-type nucleosides, cytotoxicity can be reduced and manufacturing costs can be lowered compared to ASO in which the 2' position is modified.

[0051] In this embodiment, the ASO may have some or all of the 2' positions of the nucleosides constituting the ASO modified, to the extent that it does not impair the effects of the present invention. When the 2' position of the ASO in this embodiment is modified, the modification can be appropriately selected from among the modifications commonly used in ASOs, such as 2'-MOE modification, 2'-OMe modification, and 2'-F modification.

[0052] In the ASO according to this embodiment, if a portion of all nucleosides in the ASO is modified at the 2' position, it is preferable that the 2' position of the nucleosides at the terminal end of the ASO to which the RF-aTNA structure is not bound is modified, and it is more preferable that the 2' positions of 1 to 5 nucleosides from the terminal end to which the RF-aTNA structure is not bound are modified.

[0053] In the ASO according to this embodiment, if a portion of all nucleosides in the ASO is modified at the 2' position, it is preferable that the 2' position of the nucleoside at the terminal end of the ASO to which the RF-aTNA structure is bound is unmodified, and it is more preferable that the 2' positions of 1 to 5 nucleosides from the terminal end to which the RF-aTNA structure is bound are unmodified.

[0054] The chain length of the ASO according to this embodiment is not particularly limited as long as it can function as an antisense strand, and is appropriately designed considering the base sequence of the target mRNA, etc. The chain length of the ASO according to this embodiment can be, for example, 10 to 30 bases, preferably 12 to 28 bases, and more preferably 15 to 22 bases. Furthermore, each nucleoside constituting the ASO according to this embodiment may be a deoxyribonucleoside only, a ribonucleoside only, or both a deoxyribonucleoside and a ribonucleoside.

[0055] The ASO according to this embodiment has an RF-aTNA structure at one of its two ends. In the ASO according to this embodiment, the RF-aTNA structure may be at the 5' end or the 3' end of the ASO. In the ASO of this embodiment, the RF-aTNA structure is bonded directly or via a linking group to the 5' end or 3' end of the antisense region. In the ASO according to this embodiment, it is preferable that the RF-aTNA structure is directly bonded to the 5' end or 3' end of the antisense region, and it is more preferable that the RF-aTNA structure is directly bonded to the 5' end of the antisense region.

[0056] The linking group is not particularly limited, as long as it is a group capable of linking the black circle portion of the RF-aTNA structure to the phosphate group portion at the 5' end or the ribose portion at the 3' end of the antisense region; any divalent organic group can be used. Examples of such linking groups include alkylene groups, alkenylene groups, carbonyl groups, amino groups, ether bonds, thioether bonds, ester bonds, amide bonds, polyethylene glycol groups (PEG:-(C)). ２ H ４ Examples include O)n-), siloxane bonds, silyl ether bonds, sugars, peptides, and nucleotide chains.

[0057] If the ASO according to this embodiment is a nucleic acid having structure (A1) or structure (A2) at the 5' end of the antisense region, it is preferable that the bond extending from the carbon atom at the end of structure (A1) or structure (A2) is bonded to a hydroxyl group, and the bond extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) is bonded to the sugar at the 5' end of the antisense region. If the ASO according to this embodiment is a nucleic acid having structure (A1) or structure (A2) at the 3' end of the antisense region, it is preferable that the bond extending from the carbon atom at the end of structure (A1) or structure (A2) is bonded to the phosphate group at the 3' end of the antisense region, and the bond extending from the oxygen atom of the phosphate group at the end of structure (A1) or structure (A2) is bonded to a hydrogen atom.

[0058] The ASO according to this embodiment can be produced by introducing structure (A1) or structure (A2) to one end of an oligonucleotide containing an antisense region. The oligonucleotide containing the antisense region can be produced by a conventional method using an automated nucleic acid synthesis apparatus utilizing the phosphoramidite method.

[0059] Structure (A1) or structure (A2) can be introduced into an oligonucleotide of which the structure is to be introduced by various coupling reactions. For example, by performing the phosphoramidite method using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, structure (A1) or structure (A2) can be easily introduced into either end of an oligonucleotide. Commonly used automated nucleic acid synthesizers utilize the phosphoramidite method. Therefore, by using a phosphoramidite containing structure (A1) or structure (A2) as a raw material, nucleic acids with structure (A1) or structure (A2) introduced at the desired position can be easily synthesized in an automated synthesizer for oligonucleotides of various base sequences.

[0060] Examples of phosphoramidites containing structure (A1) or structure (A2) include, among the phosphoramidites commonly used in nucleic acid synthesis, compounds in which a sugar and a phosphate group are linked via structure (A1), and compounds in which the nucleoside portion is replaced with an organic group containing structure (A2).

[0061] The synthesized target ASO can be isolated and purified by various methods, such as ion chromatography, gel filtration chromatography, reverse-phase chromatography, and normal-phase chromatography.

[0062] R ０ The group is highly hydrophobic and has high affinity for the cell membrane. Nucleic acids having structure (A1) or structure (A2) form a DNA double helix structure, R ０ The base material is exposed on the outside of the helical structure, allowing for the formation of a more stable double helix structure, and the R exposed on the surface ０ The cell membrane permeability can be improved by the group. Therefore, by making the ASO according to this embodiment a nucleic acid having structure (A1) or structure (A2), an ASO with extremely excellent cell membrane permeability can be obtained.

[0063] ASOs are generally easily degraded, so modifications at the 2'-position such as 2'-MOE modification and endosomal escape aids have been required to obtain knockdown activity. In addition, nucleic acids having an aTNA structure tend to require more time for endosomal escape compared to nucleic acids not having said structure. On the other hand, the ASO according to the present embodiment, in which an RF-aTNA structure is added to the ASO, exhibited sufficient knockdown activity even without modification at the 2'-position. Although the reason for this is not clear, R is added to an aTNA structure in ASO, which is relatively easily introduced into cells among nucleic acids. ０ The combination of the degradation-inhibiting effect resulting from introducing the group results in suppression of ASO degradation until escape from the endosome, and it is presumed that knockdown activity can be obtained even without 2'-position modification or endosomal escape aids.

[0064] In addition, the knockdown activity improving effect achieved by adding an RF-aTNA structure and PS modification is exhibited even when introduced into nucleic acids other than ASO. For example, by adding an RF-aTNA structure to any terminal of the antisense strand of siRNA and introducing one or more PS modifications, knockdown activity can be improved compared to the siRNA before introduction of these modifications.

[0065] In place of the RF-aTNA structure, even when a structure represented by the following general formula (A3) (hereinafter sometimes referred to as "structure (A3)") is added to an ASO containing a PS modification, knockdown activity can be improved compared to an ASO in which structure (A3) and PS modification are not performed.

[0066]

[0067] In general formula (A3), R ＦＥ is a C that does not have an ether-bonded oxygen atom between carbon atoms １－１０ perfluoroalkyl group, or a C having 1 to 5 ether-bonded oxygen atoms between carbon atoms ２－１０ is a perfluoroalkyl group. In general formula (A3), na is an integer of 1 to 10, n14 is an integer of 1 or more, and the solid circle represents a bonding site.

[0068] The ASO according to this embodiment can be used as is, or mixed with a pharmacologically acceptable carrier, etc., to form a pharmaceutical composition. Like other ASOs, the ASO according to this embodiment can be used as an active ingredient in pharmaceutical compositions used for the treatment or prevention of diseases in which therapeutic effects are obtained by controlling the expression of target mRNA. The ASO according to this embodiment is particularly suitable as an active ingredient in pharmaceutical compositions used in gene therapy.

[0069] When ASO according to this embodiment is used as an active ingredient in a pharmaceutical composition, ASO may be used as is, a pharmacologically acceptable salt of ASO may be used as an active ingredient, or a hydrate of ASO or a pharmacologically acceptable salt thereof may be used as an active ingredient. Examples of pharmacologically acceptable salts of ASO include alkali metal salts such as sodium salt, potassium salt, and lithium salt; alkaline earth metal salts such as calcium salt and magnesium salt; metal salts such as aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; organic amine salts such as ammonium salt, diethanolamine salt, and tetramethylammonium salt; inorganic acid salts such as nitrate, perchlorate, sulfate, and phosphate; organic acid salts such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate.

[0070] Pharmacologically acceptable carriers include various organic or inorganic carrier substances commonly used as formulation materials, for example, as excipients, lubricants, binders, and disintegrants in solid formulations; and as solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. Furthermore, formulation additives such as preservatives, antioxidants, colorants, and sweeteners may be used as needed.

[0071] The administration route of the pharmaceutical composition containing ASO as an active ingredient according to this embodiment is not particularly limited and can be appropriately determined considering the target cells, the desired therapeutic effect, etc. Examples of administration routes include oral, intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intraperitoneal, intrathecal, transrectal, transvaginal, ocular, and inhalation.

[0072] The target of administration of the pharmaceutical composition containing ASO according to this embodiment is not particularly limited and may include humans, non-human mammals, birds, reptiles, amphibians, fish, etc. Furthermore, the target of administration may also be the in vivo tissue or cultured cells of these animals.

[0073] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0074] [Example 1] ASO targeting a partial region (sense region) in the mRNA of the luciferase gene was modified, and its knockdown activity was examined.

[0075] <DNA Synthesis> In the following experiments, DNA oligonucleotides were synthesized using the phosphoramidite method with a 1 μmol scale 1000 Å dC-CPG solid support. The phosphoramidite synthesis was carried out by conventional methods using commercially available reagents, various phosphoramidites, and 5-ethylthio-1H-tetrazole as an activator.

[0076] To measure knockdown activity, an 18-nucleotide ASO (ATATCCTTGTCGTATCCC: SEQ ID NO: 1) targeting the mRNA of the luciferase gene and an oligonucleotide (MisON) (TACCCTTTTCTTACCCC: SEQ ID NO: 2) with a mismatch to the sense region in the mRNA were used.

[0077] <Synthesis of aTNA-N[FC10] and aTNA-N[FC8]> R ０ Among the groups represented by the general formula (f-1), n1 is 2, and Rf Ｐ A phosphoramidite (aTNA-N[FC10]) having a structure (A1) in which is a perfluorooctyl group, and R ０ Among the groups represented by the general formula (f-1), n1 is 2, and Rf Ｐ A phosphoramidite (aTNA-N[FC8]) having a structure (A1) in which the group is a perfluorohexyl group was synthesized by the method described in Patent Document 3. The outline of the aTNA-N[FC10] synthesis is as follows.

[0078] (1) Amide condensation

[0079]

[0080] Compound 1 (0.44 g, 0.82 mmol), PyBOP (0.43 g, 0.82 mmol), and DIPEA (0.4 mL, 2.2 equivalents), dissolved in 10 mL of DMF, were dissolved in 15 mL of DMF under argon. 4,4,5,5,6,6,7,7,8,8,9,9,10,10,11,11,11-heptadecafluoroundecanoic acid (0.23 g, 0.59 mmol, 0.7 equivalents) was added to prepare the reaction solution. The resulting reaction solution was stirred at room temperature for 14 hours. The reaction mixture was then quenched with water (45 mL) and extracted twice with hexane / ethyl acetate (4:1 (volume ratio)). The organic fractions were combined, washed with water, and Na... ２ SO ４ After drying, the product was filtered and the solvent was removed under vacuum. The resulting crude product was subjected to silica gel column chromatography (hexane:ethyl acetate:Et ３ Compound 2 was obtained by purification using N = 30:20:1 (volume ratio).

[0081] (2) Amidite formation

[0082]

[0083] 93 mg, 0.31 mmol of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorus diamidite and 40 mg, 0.31 mmol of ETT were dissolved in 8 mL of dry acetonitrile. Compound 2 (160 mg, 0.20 mmol), dissolved in a THF / acetonitrile (2 mL / 2 mL) mixed solvent, was added dropwise under argon. The reaction mixture was stirred at room temperature for 24 hours, after which the solvent was evaporated under reduced pressure. The resulting crude product was purified under argon by silica gel column chromatography using degassed hexane / ethyl acetate (3:1 (volume ratio)) as the mobile phase. Compound 3 (amiditized aTNA-N[FC10]), a colorless oil, was isolated by this method.

[0084] <(C8F17) ２- Synthesis of ASO > DNA synthesis was performed using a 1 μmol scale 1000 Å dC-CPG solid support, and two RF amidites (aTNA-N1[FC10]) were incorporated into the 5' end (C8F17). ２ - ASO was obtained on a solid phase (DMTr off). These were then cleaved from the solid support, deprotected, and purified by reverse-phase HPLC.

[0085]

[0086] <(C6F17) ２ - Synthesis of ASO > DNA synthesis was performed using a 1 μmol scale 1000 Å dC-CPG solid support, and two RF amidites (aTNA-N1[FC8]) were incorporated into the 5' end (C6F13). ２ - ASO was obtained on a solid phase (DMTr off). These were then cleaved from the solid support, deprotected, and purified by reverse-phase HPLC.

[0087]

[0088] <PS-ASO, (C8F17) ２ DNA synthesis was performed using a 1 μmol scale 1000 Å dC-CPG solid support for PS-ASO. During this process, a sulfuring agent was used during the oxidation stage of normal DNA synthesis, resulting in the synthesis of phosphorothioates in which all oxygen atoms in the phosphodiester bond sites of the native bases were replaced with sulfur atoms. This resulted in the incorporation of two RF amidites (C8F17) into the 5' end of PS-ASO (ASO in which all phosphodiester bonds are phosphorothioates) and PS-AS. ２ -PS-ASO was obtained on a solid phase (DMTr on). After cleaving and deprotecting from the solid support, impurities were removed using a simple column, and DMTr was further deprotected with 2% trifluoroacetic acid. Subsequently, it was purified by reverse-phase HPLC.

[0089] <Knockdown Assay 1 (with lipofectamine)> ASO, (C8F17) ２ -ASO, (C6F13) ２To investigate the gene silencing ability of -ASO and MisON in the presence of lipofectamine, oligonucleotides were added to luciferase-expressing human cervical adenocarcinoma (HeLa-Luc) cells, and the luciferase luminescence was evaluated by quantitative analysis.

[0090] 96-well culture plate (white plate, culture area = 0.33 cm²) ２ In a well, HeLa-Luc cells (8.0 x 10) ３ Cells (per well) are placed in Eagle's Essential Minimal Medium (EMEM, 100 μM) containing 10% fetal bovine serum (FBS) and 5% CO2. ２ The cells were incubated at 37°C for 24 hours. After culturing, the cells were washed with Dulbecco's phosphate-buffered saline (D-PBS) (100 μL x 2) and then placed in serum-free medium (Opti-MEM) supplemented with a mixture of 2 μM oligonucleotide and transfection reagent (100 μL) and 5% CO2. ２ The cells were incubated at 37°C for 24 hours. Transfection reagents used were 0.5 μL / well of "Lipofectamine 3000 Reagent" (Thermo Fisher Scientific) and 1.0 μL / well of "P3000 Reagent" (Thermo Fisher Scientific). The cell samples were then washed with D-PBS (100 μL x 2), 1×Lysis buffer (20 μL / well) was added, and the mixture was placed on an orbital shaker for 25 minutes. 100 μL of "Luciferase Assay Reagent" (manufactured by Luciferase Assay Reagent, Inc.) was added to each well, and the chemiluminescence of the cell samples was immediately measured using a plate reader (λ = 520–590 nm, n = 5). This chemiluminescence was used as an indicator of luciferase expression level.

[0091] Cells in which oligonucleotides were not added to the culture medium were used as a blank, and the inhibition rate (%) of luciferase expression in each cell sample was determined using the following formula.

[0092] [Inhibition rate (%)] = (1 - [Chemiluminescence of cells into which oligonucleotides have been introduced] / [Chemiluminescence of blank cells]) × 100%

[0093] <Knockdown Assay 2 (without lipofectamine)> 2 μM ASO, (C8F17) ２ -ASO, PS-ASO, and (C8F17) ２ To investigate the gene silencing ability of PS-ASO in the absence of lipofectamine, the amount of luciferase luminescence was evaluated by adding oligonucleotides to HeLa-Luc cells.

[0094] 96-well culture plate (white plate, culture area = 0.33 cm²) ２ In a well, HeLa-Luc cells (8.0 x 10) ３ Cells (per well) are placed in EMEM (100 μM) containing 10% FBS, and 5% CO2 ２ The cells were incubated at 37°C for 24 hours. After culturing, the cells were washed with D-PBS (100 μL x 2) and then placed in serum-free medium (Opti-MEM) supplemented with 2 μM oligonucleotides and 5% CO2. ２ The sample was incubated at 37°C for 24 hours. (C8F17) ２ -PS-ASO was added at concentrations of 100 nM, 200 nM, 500 nM, 1 μM, and 2 μM. The cell samples were then washed with D-PBS (100 μL x 2), 1×Lysis buffer (20 μL / well) was added, and the samples were placed on an orbital shaker for 25 minutes. 100 μL / well of "Luciferase Assay Reagent" (Luciferase Assay Reagent) was added, and the luminescence of the cell samples was immediately measured using a plate reader (λ = 520–590 nm, n = 5). This chemiluminescence was used as an indicator of luciferase expression. The inhibition rate (%) of luciferase expression in each cell was determined in the same manner as described above.

[0095] <Knockdown Assay 1 (with lipofectamine)> 2 μM ASO, (C8F17) ２ - ASO, and (C6F13) ２- The results of the luciferase assay of ASO in the presence of lipofectamine are shown in Table 1. In Table 1, "×" in the "Inhibition Rate [%]" column means that the chemiluminescence level was equal to or greater than that of the blank, and that no inhibition of luciferase expression by the added oligonucleotide was confirmed.

[0096] No inhibition of luciferase expression was observed in cells introduced with MisON. On the other hand, as shown in Table 1, ASO, (C8F17) ２ - ASO, and (C6F13) ２ - In ASO, inhibition of luciferase expression was observed in 28%, 15%, and 17% of cases, respectively. Since MisON did not inhibit luciferase expression, ASO and (C8F17) ２ - ASO, and (C6F13) ２ - The inhibition of luciferase expression by ASO was attributed to the recognition and binding of the luciferase sequence by the antisense nucleic acid. Compared to ASO, (C8F17) ２ - ASO and (C6F13) ２ - The reason why the inhibition rate of luciferase expression in ASO was low is C ８ F １７ Base and C ６ F １３ It was considered possible that aggregate formation by the group hindered the efficient encapsulation of antisense nucleic acids by lipofectamine, or inhibited the proper recognition of the luciferase sequence. On the other hand, (C8F17) ２ - ASO and (C6F13) ２ - No difference in luciferase expression inhibition rates was observed among ASOs due to differences in RF group length.

[0097] <Knockdown Assay 2 (without lipofectamine)> 2 μM ASO, (C8F17) ２ -ASO, PS-ASO, and (C8F17) ２ Tables 1 and 2 show the results of the luciferase assay of PS-ASO in the absence of lipofectamine. In Table 2, the "×" in the "Inhibition Rate [%]" column is the same as in Table 1.

[0098] ASO, (C8F17) ２-ASO and PS-ASO did not inhibit luciferase expression. On the other hand, (C8F17) ２ -PS-ASO showed a 21% inhibition of luciferase expression. This is (C8F17) ２ It was hypothesized that luciferase expression was inhibited because PS-ASO achieved highly efficient intracellular uptake and endosomal exit. Furthermore, 100 nM, 200 nM, 500 nM, and 1 μM (C8F17) ２ Table 2 shows the results of the luciferase assay of PS-ASO in the absence of lipofectamine. (C8F17) ２ PS-ASO inhibited luciferase expression by 3%, 18%, 20%, 17%, and 21%, respectively. Since the inhibition rate did not change at concentrations above 200 nM, (C8F17) ２ It was estimated that the maximum inhibition rate of luciferase expression by PS-ASO is approximately 20%.

[0099]

[0100]

[0101] [Reference Example 1] R in oligonucleotide ０ Among the groups represented by the general formula (f-1), n1 is 2, and Rf Ｐ We added a structure (A1) in which a perfluorooctyl group was attached and investigated its cell membrane permeability.

[0102] Using TNA-N[FC10], an oligonucleotide (control DNA) consisting of the base sequence of SEQ ID NO: 3 (TTTTTCAGTTGACCATATA) had 2 to 5 RF amidites (aTNA-N1[FC10]) incorporated at its 5' end, and fluorescein was added to its 3' end (C8F17). ２ -DNA-FAM, (C8F17) ３ -DNA-FAM, (C8F17) ４ - DNA-FAM, and (C8F17) ５ DNA-FAM was synthesized. As a control, DNA-FAM was also synthesized by adding fluorescein to the 3' end of control DNA.

[0103] <Preparation of Sample Solutions> Each FAM-modified oligonucleotide (DNA-FAM and (C8F17)n-DNA-FAM (n=2-5)) was prepared in Dulbecco's modified Eagle medium (DMEM) to a concentration of 2.0 μM, and these were used as sample solutions. A 2.0 μM solution of DNA-FAM was used as the negative control. DMEM was used for blank measurements.

[0104] <Evaluation of cell membrane permeability by flow cytometry> HeLa cells (1.0 × 10⁻⁶) ４ Cells / well, 96-well culture plate, culture area = 0.33 cm² ２ ( / well) in DMEM (100 μM) containing 10% FBS, 5% CO2 ２ The cells were incubated at 37°C for 24 hours. Then, the cells were washed with D-PBS (100 μL x 2), and serum-free DMEM (100 μL) containing 2 μM FAM-modified oligonucleotides was added, followed by 5% CO2. ２ The cells were incubated at 37°C for 4 hours. Afterward, the cell samples were washed with D-PBS (100 μL) and mixed with EDTA-trypsin (10 μM) and 5% CO2. ２ The cells were incubated at 37°C for 3 minutes. Next, the cell sample was diluted with DMEM (200 μL), and the resulting suspension was centrifuged at 1200 rpm for 3 minutes. After removing the supernatant, the resulting cell pellet was suspended in D-PBS (250 μL), and the intracellular fluorescence intensity was quantified using a flow cytometer (Luminex model Guava easyCyto®) (excitation wavelength = 488 nm, detection fluorescence wavelength = 510–540 nm, n = 3).

[0105] Cells in which no FAM-modified oligonucleotides were added to the culture medium were used as a blank, and cells in which only DNA-FAM was added to the culture medium were used as a negative control. Fluorescence intensity indicates the amount of each FAM-modified oligonucleotide taken up by the cells. The relative fluorescence intensity of each cell sample to the blank fluorescence intensity was calculated. The results are shown in Table 3.

[0106]

[0107] As shown in Table 3, three units of structure (A1) were connected to form (C8F17). ３ - The fluorescence intensity of cells into which DNA-FAM was introduced was more than 150 times higher than that of cells into which DNA-FAM was introduced, indicating the highest level of cellular uptake. On the other hand, (C8F17) ４ - The amount of DNA-FAM uptake in cells introduced decreased to about 10%. (C8F17) ５ - Although it improved again with DNA-FAM, (C8F17) ２ - The amount of DNA-FAM taken up by cells was about half of what it was.

[0108] [Example 2] An ASO targeting a partial region (sense region) of the luciferase gene mRNA was modified with phosphorothioate binding and an RF-aTNA structure, and its knockdown activity was examined.

[0109] The modified ASO used was (C8F17) synthesized in Example 1. ２ - In addition to PS-ASO, (C8F17) ２ In the 3'-terminus of PS-ASO, five oxygen atoms in the phosphodiester bond sites were replaced with sulfur atoms (C8F17). ２ -PS(3') / PO-ASO and (C8F17) ２ - Ten oxygen atoms in the phosphodiester bond sites of ASO (five from the 5' end and five from the 3' end) were replaced with sulfur atoms (C8F17). ２ -PS(3',5') / PO-ASO was used.

[0110] <(C8F17) ２ -PS(3') / PO-ASO and (C8F17) ２ Oligonucleotide synthesis was performed using a 1 μmol scale 1000 Å dC-CPG solid support with the formula -PS(3',5') / PO-ASO>. In this process, a surfuring agent was used during a portion of the oxidation process in the normal oligonucleotide synthesis, and RF amidite (aTNA-N1[FC10]) was appropriately used as a starting material, resulting in (C8F17). ２ -PS(3') / PO-ASO and (C8F17) ２-PS(3',5') / PO-ASO were obtained on the solid phase (DMTr on). After cleaving and deprotecting these from the solid support, impurities were removed using a simple column, and DMTr was further deprotected with 2% trifluoroacetic acid. Subsequently, the samples were purified by reverse-phase HPLC.

[0111] <Knockdown Assay> (C8F17) ２ -PS-ASO, (C8F17) ２ -PS(3') / PO-ASO, and (C8F17) ２ To investigate the gene silencing ability of -PS(3',5') / PO-ASO in the absence of lipofectamine, the enzyme was evaluated by adding oligonucleotides to luciferase-expressing human cervical adenocarcinoma (HeLa-Luc) cells and quantifying the amount of luciferase luminescence.

[0112] 96-well culture plate (white plate, culture area = 0.33 cm²) ２ In a well, HeLa-Luc cells (5.0 × 10) ３ Cells (per well) are placed in EMEM (100 μM) containing 10% FBS, and 5% CO2 ２ The cells were incubated at 37°C for 24 hours. After culturing, the cells were washed with D-PBS (100 μL x 2). Then, they were incubated in serum-free medium (Opti-MEM) supplemented with 2 μM oligonucleotides and 5% CO2. ２ The cells were incubated at 37°C for 24 hours. Afterward, the cell samples were washed with D-PBS (100 μL x 2), 1×Lysis buffer (20 μL / well) was added, and the mixture was shaken. Next, a luciferase assay was performed using "Luciferase Assay Reagent" (manufactured by Luciferase Assay Reagent) in the same manner as in Example 1's <Knockdown Assay 1 (with lipofectamine)>, and the inhibition rate (%) of luciferase expression in each cell was determined. The results are shown in Table 4.

[0113]

[0114] As shown in Table 4, all phosphodiester bonds were replaced with phosphorothioate bonds (C8F17).２ While the inhibition rate of PS-ASO was 25%, the phosphodiester bond at the 3' end of ASO was replaced with a phosphorothioate bond (C8F17). ２ The inhibition rate of -PS(3') / PO-ASO was 21%, (C8F17) ２ -PS-ASO was comparable in performance. On the other hand, the phosphodiester bonds at both ends of ASO were replaced with phosphorothioate bonds (C8F17). ２ The inhibition rate for -PS(3',5') / PO-ASO was 40%, which was the highest inhibition rate.

[0115] Because the antisense oligonucleotide according to this embodiment exhibits excellent knockdown efficiency of target genes, it is expected to be used in the pharmaceutical field, for example, as an active ingredient in nucleic acid drugs.

Claims

1. An antisense oligonucleotide having one or more phosphorothioate bonds, wherein one of the ends of the antisense oligonucleotide is a compound of the following general formula (A1) or (A2) [In the formula, R ０ An antisense oligonucleotide having an aTNA structure represented by [where n11 and n12 are each independently integers of 1 or more; B is a nucleic acid base; and black circles indicate bonding].

2. The antisense oligonucleotide according to claim 1, wherein at least one of the bonds between the 1 to 5 nucleotides at the terminal end of the antisense oligonucleotide that is not bound to the aTNA structure is a phosphorothioate bond.

3. The antisense oligonucleotide according to claim 1, wherein the antisense oligonucleotide has one or more phosphodiester bonds.

4. The antisense oligonucleotide according to claim 1, wherein all bonds between the nucleotides constituting the antisense oligonucleotide are phosphorothioate bonds.

5. The antisense oligonucleotide according to claim 1, wherein the 2' position of deoxyribose or ribose in all nucleosides constituting the antisense oligonucleotide is unmodified.

6. The antisense oligonucleotide according to claim 1, wherein n11 or n12 is 2 or more.

7. The antisense oligonucleotide according to claim 1, wherein n11 or n12 is 2 or 3.

8. The aforementioned R ０ The antisense oligonucleotide according to claim 1, wherein the alkyl group has 1 to 30 carbon atoms and is substituted with at least two fluorine atoms.

9. The aforementioned R ０ The antisense oligonucleotide according to claim 1, wherein the group is a perfluoroalkyl group having 1 to 10 carbon atoms, or a perfluoroalkyl group having 2 to 10 carbon atoms with 1 to 5 ether-bonded oxygen atoms between the carbon atoms.

10. The aforementioned R ０ The antisense oligonucleotide according to claim 1, wherein the group is an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom, or a group having 1 to 5 ether-bonded oxygen atoms between the carbon atoms of an alkyl group having 10 to 30 carbon atoms that is not substituted with a fluorine atom.

11. A pharmaceutical composition comprising an antisense oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 10, or a hydrate thereof.

12. The pharmaceutical composition according to claim 11, used in gene therapy.

13. Use of an antisense oligonucleotide or a pharmaceutically acceptable salt thereof, or a hydrate thereof, according to any one of claims 1 to 10, for the manufacture of a gene therapy agent.

Images