阅读说明：本技术 使用经结构强化的S-TuD的新的癌症治疗方法 (Novel cancer treatment methods using structurally enhanced S-TuD ) 是由 伊庭英夫 原口健 南海浩一 佐藤秀昭 于 2018-03-16 设计创作，主要内容包括：提供使用经结构强化的S-TuD的新的癌症治疗方法。提供一种组合物,其用于预防或处置肿瘤且包含含有RNA或其类似物的miRNA抑制复合体,或者提供使用该组合物的用于预防或处置肿瘤的方法。miRNA抑制复合体优选包含至少1个双链结构和miRNA结合序列。优选miRNA结合序列的两条链分别与双链结构的至少一端的两条链中的1条结合。本发明的部分方面还提供用于递送这种miRNA抑制复合体的递送系统。(Novel cancer treatment methods using structurally-enhanced S-TuD are provided. Provided is a composition for preventing or treating a tumor comprising a miRNA-inhibiting complex comprising an RNA or an analog thereof, or a method for preventing or treating a tumor using the composition. The miRNA inhibition complex preferably comprises at least 1 double stranded structure and a miRNA binding sequence. Preferably, both strands of the miRNA binding sequence bind to 1 of the two strands at least one end of the double stranded structure. Aspects of the invention also provide delivery systems for delivering such miRNA-inhibitory complexes.)

1. A composition for use in the prevention or treatment of a tumor comprising a miRNA-inhibition complex comprising RNA or an analog thereof, the miRNA-inhibition complex comprising at least 1 double-stranded structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the double-stranded structure, respectively, the miRNA-inhibition complex comprising at least 1 Bridging Nucleic Acid (BNA).

2. The composition of claim 1, wherein said BNA comprises a compound of the structure or a 2', 4' methylene bridged nucleic acid (LNA),

[ solution 47]

3. The composition according to claim 1, wherein said BNA is BNA^NC(NMe)。

4. The composition according to any one of claims 1 to 3, wherein the miRNA-inhibiting complex comprises 2 or more double-stranded structures, wherein one miRNA-binding sequence-containing strand is bound to each of two strands at one end of a first double-stranded structure of the double-stranded structures, and wherein the other end of each strand is bound to each of two strands of a second double-stranded structure of the 2 or more double-stranded structures, whereby the strands are sandwiched between the 2 or more double-stranded structures.

5. The composition of claim 4, wherein the miRNA-inhibiting complex comprises 2 miRNA binding sequences.

6. The composition of claim 4, wherein the miRNA inhibition complex comprises a structure shown below,

[ solution 48]

(C)

The structure I and II are double-stranded structures, and the structure a and b respectively contain 1 miRNA binding sequence.

7. The composition according to any one of claims 1 to 6, wherein the miRNA-inhibiting complex comprises 2 miRNA binding sequences, one miRNA binding sequence comprising 5'-CAGUGUU-3' and the other miRNA binding sequence comprising 5 '-CAGUAUU-3'.

8. A nucleic acid molecule comprising 2 miRNA-binding sequences, wherein one miRNA-binding sequence comprises 5'-CAGUGUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the other miRNA-binding sequence comprises 5'-CAGUAUU-3' (in the sequence, uracil bases are thymine bases if necessary), and wherein the nucleic acid molecule comprises at least 1 Bridging Nucleic Acid (BNA).

9. A nucleic acid molecule comprising a miRNA-binding sequence comprising the sequence of seq id No. 1 (in the sequence, uracil bases are thymine bases as appropriate), and a miRNA-binding sequence comprising the sequence of seq id No. 2, and said nucleic acid molecule comprising at least 1 Bridging Nucleic Acid (BNA).

10. A nucleic acid molecule comprising the sequence of SEQ ID NO. 9 (in the sequence, uracil base is thymine base if necessary) and the sequence of SEQ ID NO. 10 (in the sequence, uracil base is thymine base if necessary).

11. A nucleic acid molecule comprising a sequence of 5'-AUAAGCU-3' (in the sequence uracil bases are optionally thymine bases) and comprising at least 1 Bridging Nucleic Acid (BNA).

12. A composition comprising the nucleic acid molecule of any one of claims 8-11.

13. The composition according to claim 12 for use in the prevention or treatment of tumors.

14. The composition of any one of claims 1 to 7 or 13, wherein the tumor is a carcinoma.

15. The composition of any one of claims 1 to 7 or 13, wherein the tumor is colon cancer, lung cancer or breast cancer.

16. The composition of any one of claims 1-7 or 13-15 for use in promoting epithelial-mesenchymal transition of the tumor.

17. The composition of any one of claims 1 to 7 or 12 to 16, wherein the miRNA inhibitory complex or nucleic acid molecule is present in a form comprised in a vector for nucleic acid delivery.

18. A composition comprising a miRNA-inhibition complex comprising RNA or an analog thereof, the miRNA-inhibition complex comprising at least 1 double-stranded structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the double-stranded structure, respectively, and a carrier for nucleic acid delivery, the miRNA-inhibition complex comprising at least 1 Bridging Nucleic Acid (BNA).

19. A composition comprising a lipid membrane structure and a nucleic acid complex encapsulated by the lipid membrane structure, the lipid membrane structure comprising a compound represented by formula (1) as a constituent lipid of a membrane,

[ solution 49]

In the formula (1), X^aAnd X^bAre each independently X¹、X²Or X³；

[ solution 50]

s is a number of 1 or 2,

R⁴represents an alkyl group having 1 to 6 carbon atoms,

n^aand n^bAre each independently 0 or 1, respectively,

R^1aand R^1bEach independently represents an alkylene group having 1 to 6 carbon atoms,

R^2aand R^2bEach independently represents an alkylene group having 1 to 6 carbon atoms,

Y^aand Y^bEach independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond,

R^3aand R^3bIndependently represents a sterol residue, a fat-soluble vitamin derivative residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms,

the sterol residue is cholesteryl, cholestanyl, stigmasteryl, beta-sitsteryl, lanosterol or ergosteryl,

the fat-soluble vitamin is retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, vitamin D2, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol or tocotrienol,

the nucleic acid complex is a miRNA-inhibiting complex comprising RNA or an analog thereof, the miRNA-inhibiting complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the duplex structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

20. The composition according to claim 19, wherein, in formula (1), X^aAnd X^bIndependently is X²。

21. The composition according to claim 19, wherein, in formula (1), X^aAnd X^bIndependently is X³。

22. The composition according to any one of claims 19 to 21, wherein in the formula (1), R is^3aAnd R^3bIndependently a fat-soluble vitamin derivative residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

23. A composition according to any one of claims 19 to 22, wherein R is^3aAnd R^3bIndependently a fat-soluble vitamin derivative residue.

24. The composition of claim 23, wherein the fat-soluble vitamin derivative residue is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride.

25. The composition of any one of claims 19-24, wherein R^3aAnd R^3bIndependently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

26. The composition of any one of claims 19-25, wherein said BNA is BNA^NC(NMe)。

27. The composition according to any one of claims 19 to 26, wherein the miRNA inhibition complex comprises 2 or more double-stranded structures, one strand each comprising a miRNA binding sequence is bound to each of two strands at one end of a first double-stranded structure of the double-stranded structures, and each other end of the strand is bound to each of two strands of a second double-stranded structure of the 2 or more double-stranded structures, whereby the strand is sandwiched by the 2 or more double-stranded structures.

28. The composition of claim 26, wherein the miRNA-inhibitory complex comprises 2 miRNA-binding sequences.

29. The composition of claim 27, wherein the miRNA-inhibiting complex comprises a structure shown below,

[ solution 51]

(C)

The structure I and II are double-stranded structures, and the structure a and b respectively contain 1 miRNA binding sequence.

30. The composition according to any one of claims 19 to 29, wherein the miRNA-inhibitory complex comprises 2 miRNA-binding sequences, one of the miRNA-binding sequences comprises 5 '-cagugu-3' (in which uracil bases are thymine bases if necessary), and the other of the miRNA-binding sequences comprises 5'-CAGUAUU-3' (in which uracil bases are thymine bases if necessary).

31. The composition according to any one of claims 19 to 30, wherein the miRNA-inhibitory complex comprises a miRNA binding sequence comprising the sequence of seq id No. 1 (uracil base is thymine base if necessary in the sequence) and a miRNA binding sequence comprising the sequence of seq id No. 2 (uracil base is thymine base if necessary in the sequence).

32. The composition according to any one of claims 19 to 31, wherein the miRNA-inhibitory complex comprises the sequence of seq id No. 9 (uracil base is thymine base if necessary in the sequence) and the sequence of seq id No. 10 (uracil base is thymine base if necessary in the sequence).

Technical Field

The present invention relates to novel cancer treatment methods, related therapeutic agents, drug delivery vehicles using structurally-strengthened synthetic strong baits (S-TuD).

Background

Micro rna (miRNA) plays an important role in a variety of life phenomena including occurrence by controlling a plurality of target genes to form a gene regulatory network, and various inhibitors for inhibiting miRNA have been developed (WO2010/047216 ═ patent document 1).

In addition, some of the present inventors have been developing enhanced inhibitory nucleic acids using inhibitory nucleic acids such as S-TuD for experimental miRNA inhibition.

Disclosure of Invention

Means for solving the problems

The present inventors have found that when at least 1 Bridge Nucleic Acid (BNA) is contained in a miRNA inhibitory complex containing RNA or an analog thereof (in the present specification, a Nucleic Acid containing BNA is also referred to as a BNA-modified inhibitory Nucleic Acid "S-TuD"), the inhibitory activity is consolidated, the biological activity is enhanced, and a disease (for example, cancer) can be effectively treated, and thus the present invention has been completed. In particular embodiments, the present inventors have found that tumors can be effectively inhibited by using a complex comprising at least 1 Bridging Nucleic Acid (BNA) in the inhibition of cancer-associated mirnas (e.g., the miR-200 family). It was also found that this effect was also observed in the simultaneous inhibition of multiple different members of cancer-associated mirnas.

In the invention, the BNA modified inhibitory nucleic acid S-TuD is found to be miniaturized, have increased serum stability and enhanced microRNA inhibitory capacity compared with the traditional S-TuD, thereby realizing a tumor treatment method based on miRNA inhibition. The BNA-modified inhibitory nucleic acid "S-TuD" of the present invention is expected to be improved more effectively than the conventional S-TuD.

Thus, in various embodiments, an embodiment of the invention provides a composition for the prevention or treatment of a tumor comprising a miRNA-inhibitory complex comprising RNA or an analog thereof and comprising at least 1 BNA; or a method for preventing or treating a tumor using the composition. The miRNA inhibitory complex comprises at least 1 double-stranded structure and a miRNA binding sequence. Two strands of the miRNA binding sequence bind to 1 of the two strands at least one end of the double-stranded structure. By adopting such a structure, the miRNA inhibitory complex used in the present invention has increased serum stability and enhanced miRNA inhibitory ability, and is advantageous for the prevention or treatment of tumors.

For example, preferred embodiments of the present invention provide the following items.

(item a1) a composition for use in the prevention or treatment of a tumor comprising a miRNA-inhibition complex comprising RNA or an analog thereof, the miRNA-inhibition complex comprising at least 1 double-stranded structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the double-stranded structure, respectively, the miRNA-inhibition complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item a2) the composition of the above item, wherein the BNA comprises a BNA bridged via at least 1 atom on the 2 'side selected from the group consisting of oxygen and carbon, via carbon on the 4' side, and at least 1 atom selected from the group consisting of carbon and nitrogen.

(item A3) the composition of any one of the above items, wherein the BNA comprises a2 ', 4' substituted bridging nucleic acid represented by the following formula (BNA-1),

[ solution 1]

(in the formula (BNA-1), R₁、R_1’、R₂、R_2’And R₃Each independently represents a group selected from the group consisting of a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted acyl group, a substituted or unsubstituted sulfonyl group, a substituted or unsubstituted silyl group, and a functional molecular unit substituent, m is an integer of 0 to 2, Base represents a group selected from the group consisting of an adenine group, a thymine group, a uracil group, a inosine group, a cytosine group, a guanidino group, and a methylcytosine group, n is an integer of 1 to 3, and q is an integer of 0 or 1. ).

(item A4) the composition of any one of the above items, wherein the BNA comprises a2 ', 4' -substituted bridging nucleic acid represented by the following formula (BNA-2),

[ solution 2]

(in the formula (BNA-2), R₃Represents a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, and a functional molecular unit substituent, Base represents a group selected from the group consisting of an adenine group, a thymine group, a uracil group, a inosine group, a cytosine group, a guanidinium group, and a methylcytosine group, m is an integer of 0 to 2, and n is an integer of 1 to 3. ).

(item A5) the composition of any one of the above items, wherein the BNA comprises a compound represented by the formula or a2 ', 4' methylene bridged nucleic acid (LNA),

[ solution 3]

(item A6) the composition of any one of the above items, wherein the BNA is BNA^NC(NMe)。

(item a7) the composition according to any one of the above items, wherein the miRNA inhibition complex comprises 2 or more of the double-stranded structures, one strand each comprising a miRNA binding sequence is bound to each of two strands at one end of a first double-stranded structure of the double-stranded structures, and each other end of the strand is bound to each of two strands of a second double-stranded structure of the 2 or more double-stranded structures, whereby the strand is sandwiched by the 2 or more double-stranded structures.

(item A8) the composition of any one of the above items, wherein the double-stranded structure is at least 6 bases long.

(item A9) the composition of any one of the above items, wherein the double-stranded structure is at least 8 bases long.

(item A10) the composition according to any one of the above items, wherein the double-stranded structure has a length of 50 nucleotides or less.

(item a11) the composition of any one of the above items, wherein the miRNA inhibition complex comprises 2 to 5 miRNA binding sequences.

(item a12) the composition of any one of the above items, wherein the miRNA inhibition complex comprises 2 miRNA binding sequences.

(item A13) the composition according to any one of the above items, wherein the miRNA inhibitory complex comprises a structure shown below,

[ solution 4]

The structure I and II are double-stranded structures, and the structure a and b respectively contain 1 miRNA binding sequence.

(item A14) the composition according to any one of the above items, wherein the miRNA binding sequence comprises 5'-CAGUGUU-3' (in the sequence, the uracil base is a thymine base if necessary) and/or 5'-CAGUAUU-3' (in the sequence, the uracil base is a thymine base if necessary).

(item A15) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises 2 miRNA binding sequences, one of the miRNA binding sequences comprises 5'-CAGUGUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the other of the miRNA binding sequences comprises 5'-CAGUAUU-3' (in the sequence, uracil bases are thymine bases if necessary).

(item A16) A nucleic acid molecule comprising a sequence of 5'-CAGUGUU-3' (in the sequence, the uracil base is optionally a thymine base) and 5'-CAGUAUU-3' (in the sequence, the uracil base is optionally a thymine base), said nucleic acid molecule comprising at least 1 Bridging Nucleic Acid (BNA).

(item A16-2) A nucleic acid molecule comprising 2 miRNA-binding sequences, wherein one miRNA-binding sequence comprises 5'-CAGUGUU-3' (in the sequence, the uracil base is a thymine base if necessary) or 5'-CAGUAUU-3' (in the sequence, the uracil base is a thymine base if necessary), and the other miRNA-binding sequence comprises 5 '-CAGUU-3' (in the sequence, the uracil base is a thymine base if necessary) or 5'-CAGUAUU-3' (in the sequence, the uracil base is a thymine base if necessary), and the nucleic acid molecule comprises at least 1 Bridging Nucleic Acid (BNA).

(item A17) A nucleic acid molecule comprising 2 miRNA-binding sequences, wherein one miRNA-binding sequence comprises 5'-CAGUGUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the other miRNA-binding sequence comprises 5'-CAGUAUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the nucleic acid molecule comprises at least 1 Bridging Nucleic Acid (BNA).

(item A18A) A nucleic acid molecule comprising a miRNA-binding sequence comprising the sequence of SEQ ID NO. 1 (in the sequence, the uracil base is optionally a thymine base) and a miRNA-binding sequence comprising the sequence of SEQ ID NO. 2 (in the sequence, the uracil base is optionally a thymine base).

(item A18B) A nucleic acid molecule comprising a miRNA-binding sequence comprising the sequence of SEQ ID NO. 3 (in the sequence, the uracil base is optionally a thymine base) and a miRNA-binding sequence comprising the sequence of SEQ ID NO. 4 (in the sequence, the uracil base is optionally a thymine base).

(item A19A) A nucleic acid molecule comprising the sequence of SEQ ID NO. 9 (in the sequence, the uracil base is a thymine base as required) and the sequence of SEQ ID NO. 10 (in the sequence, the uracil base is a thymine base as required), said nucleic acid molecule comprising at least 1 Bridging Nucleic Acid (BNA).

(item A19B) A nucleic acid molecule comprising the sequence of SEQ ID NO. 5 (in the sequence, the uracil base is a thymine base if necessary) and the sequence of SEQ ID NO. 6 (in the sequence, the uracil base is a thymine base if necessary).

(item AA1) A nucleic acid molecule comprising a sequence of 5'-AUAAGCU-3' (in which the uracil base is optionally a thymine base), said nucleic acid molecule comprising at least 1 Bridging Nucleic Acid (BNA).

(item AA2) A nucleic acid molecule comprising a miRNA-binding sequence comprising the sequence of SEQ ID NO. 33 (in which sequence the uracil bases are optionally thymine bases), said nucleic acid molecule comprising at least 1 Bridging Nucleic Acid (BNA).

(item AA3) A nucleic acid molecule comprising a miRNA binding sequence comprising the sequence of SEQ ID NO. 34 (wherein the uracil base is optionally a thymine base).

(item AA4) A nucleic acid molecule comprising the sequence of SEQ ID NO. 37 and/or SEQ ID NO. 38.

(item a20) a composition comprising the nucleic acid molecule of any one of the above items.

(item a21) the composition according to any one of the above items, for use in the prevention or treatment of a tumor.

(item A22) the composition according to any one of the above items, wherein the tumor is cancer (carcinoma).

(item A23) the composition according to any one of the above items, wherein the tumor is colon cancer (colon cancer), lung cancer (lung cancer) or breast cancer (breast cancer).

(item a24) the composition of any one of the above items for use in promoting epithelial-mesenchymal transition of the above tumor.

(item a25) the composition of any one of the above items, wherein the miRNA inhibitory complex or the nucleic acid molecule is present in a form contained in a vector for nucleic acid delivery.

(item a26) the composition of any one of the above items, wherein the carrier is selected from the group consisting of Lipid Nanoparticles (LNPs), cationic liposomes, non-cationic liposomes, cationic polymers, non-cationic polymers, beta glucans, atelocollagen, PLGA nanoparticles, surfactant peptides and super apatites.

(item a27) the composition of any one of the above items, wherein the carrier is LNP, and the LNP comprises a cationic lipid.

(item a28) the composition of any one of the above items, wherein said LNP comprises a cationic lipid, a helper lipid, and a PEG-modified lipid.

(item a29) the composition according to any one of the above items, wherein the cationic lipid contains a tertiary amine and/or a disulfide bond in a molecule.

(item B1) a composition comprising a miRNA-inhibition complex comprising RNA or an analog thereof, the miRNA-inhibition complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding 1 each to two strands at least one end of the duplex structure, the miRNA-inhibition complex comprising at least 1 Bridging Nucleic Acid (BNA), and a carrier for nucleic acid delivery.

(item B1-1) the composition of item B1 above, having the features of any one of items A1 to A29.

(item B2) the composition of any one of the above items, which is a pharmaceutical composition.

(item B3) the composition of any one of the above items for use in delivering the miRNA-inhibitory complex to a desired site.

(item B4) the composition of any one of the above items, wherein the BNA comprises a BNA bridged via at least 1 atom from the group consisting of oxygen and carbon on the 2 'side, carbon on the 4' side, and at least 1 atom from the group consisting of carbon and nitrogen.

(item B5) the composition of any one of the above items, wherein the BNA comprises a2 ', 4' -substituted bridging nucleic acid represented by the following formula (BNA-1),

[ solution 5]

(in the formula (BNA-1), R₁、R_1’、R₂、R_2’And R₃Each independently represents a hydrogen atom, substituted orAn unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted acyl group, a substituted or unsubstituted sulfonyl group, a substituted or unsubstituted silyl group, and a functional molecular unit substituent, m is an integer of 0 to 2, Base represents a group selected from the group consisting of an adenine group, a thymine group, a uracil group, a inosine group, a cytosine group, a guanidinium group, and a methylcytosine group, n is an integer of 1 to 3, and q is an integer of 0 or 1. ).

(item B6) the composition of any one of the above items, wherein the BNA comprises a2 ', 4' -substituted bridging nucleic acid represented by the following formula (BNA-2),

[ solution 6]

(in the formula (BNA-2), R₃Represents a group selected from the group consisting of a hydrogen atom, an alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a sulfonyl group, a silyl group, and a functional molecular unit substituent, Base represents a group selected from the group consisting of an adenine group, a thymine group, a uracil group, a inosine group, a cytosine group, a guanidinium group, and a methylcytosine group, m is an integer of 0 to 2, and n is an integer of 1 to 3. ).

(item B7) the composition of any one of the above items, wherein the BNA comprises a compound of the structure or a2 ', 4' methylene bridged nucleic acid (LNA),

[ solution 7]

(item B8) the composition of any one of the above items, wherein the BNA is BNA^NC(NMe)。

(item B9) the composition according to any one of the above items, wherein the miRNA inhibition complex comprises 2 or more of the double-stranded structures, one strand each comprising a miRNA binding sequence is bound to each of two strands at one end of a first double-stranded structure of the double-stranded structures, and each of the other ends of the strands is bound to each of two strands of a second double-stranded structure of the 2 or more double-stranded structures, and is sandwiched between the 2 or more double-stranded structures.

(item B10) the composition of any one of the above items, wherein the double-stranded structure is at least 6 bases long.

(item B11) the composition of any one of the above items, wherein the double-stranded structure is at least 8 bases long.

(item B12) the composition according to any one of the above items, wherein the double-stranded structure has a length of 50 nucleotides or less.

(item B13) the composition of any one of the above items, wherein the miRNA inhibition complex comprises 2 to 5 miRNA binding sequences.

(item B14) the composition of any one of the above items, wherein the miRNA inhibition complex comprises 2 miRNA binding sequences.

(item B15) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a structure shown below,

[ solution 8]

The structure I and II are double-stranded structures, and the structure a and b respectively contain 1 miRNA binding sequence.

(item B16) the composition of any one of the above items, wherein the miRNA binding sequence comprises 5'-CAGUGUU-3' (the uracil base is a thymine base if necessary in the sequence) and/or 5'-CAGUAUU-3' (the uracil base is a thymine base if necessary in the sequence).

(item B17) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises 2 miRNA binding sequences, one of the miRNA binding sequences comprises 5'-CAGUGUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the other of the miRNA binding sequences comprises 5'-CAGUAUU-3' (in the sequence, uracil bases are thymine bases if necessary).

(item B18A) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a miRNA-binding sequence of SEQ ID NO. 1 (uracil base, if necessary, thymine base in the sequence) and a miRNA-binding sequence of SEQ ID NO. 2 (uracil base, if necessary, thymine base in the sequence).

(item B18B) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a miRNA-binding sequence of SEQ ID NO. 3 (uracil base, if necessary, thymine base in the sequence) and a miRNA-binding sequence of SEQ ID NO. 4 (uracil base, if necessary, thymine base in the sequence).

(item B19A) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises the sequence of SEQ ID NO. 5 (in which the uracil base is a thymine base if necessary) and the sequence of SEQ ID NO. 6 (in which the uracil base is a thymine base if necessary).

(item B19B) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises the sequence of SEQ ID NO. 9 (in which the uracil base is a thymine base if necessary) and the sequence of SEQ ID NO. 10 (in which the uracil base is a thymine base if necessary).

(item B20) the composition of any one of the above items, wherein the carrier is selected from the group consisting of Lipid Nanoparticles (LNPs), cationic liposomes, non-cationic liposomes, cationic polymers, non-cationic polymers, beta glucans, atelocollagen, PLGA nanoparticles, surfactant peptides and super apatites.

(item B21) the composition of any one of the above items, wherein the carrier is LNP, and the LNP comprises a cationic lipid.

(item B22) the composition of any one of the above items, wherein the LNP comprises a cationic lipid, a helper lipid, and a PEG-modified lipid.

(item B23) the composition according to any one of the above items, wherein the cationic lipid contains a tertiary amine and/or a disulfide bond in a molecule.

(item C1) A composition which is a composition comprising a lipid membrane structure comprising a compound represented by the formula (1') as a constituent lipid of a membrane, and a nucleic acid complex encapsulated by the lipid membrane structure,

[ solution 9]

(in the formula (1'),

X^aand X^bEach independently a tertiary amine-containing substituent,

s is a number of 1 or 2,

R⁴represents an alkyl group having 1 to 6 carbon atoms,

n^aand n^bAre each independently 0 or 1, respectively,

R^1aand R^1bEach independently represents an alkylene group having 1 to 6 carbon atoms,

R^2aand R^2bEach independently represents an alkylene group having 1 to 6 carbon atoms,

Y^aand Y^bEach independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond,

R^3aand R^3bIndependently represents a sterol residue, a fat-soluble vitamin derivative residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms,

the sterol residue is cholesteryl, cholestanyl, stigmasteryl, beta-sitsteryl, lanosterol or ergosteryl,

the fat-soluble vitamin is retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, vitamin D2, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol or tocotrienol),

the nucleic acid complex is a miRNA-inhibiting complex comprising RNA or an analog thereof, the miRNA-inhibiting complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the duplex structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item C1-1) the composition of item C1, having the features of any one of items A1-29 or items B1-23.

(item C2) the composition of any one of the above items, wherein X^aAnd X^bAre each independently X¹、X²Or X³；

[ solution 10]

(item C3) the composition according to any one of the above items, wherein, in formula (1'), R^3aAnd R^3bIndependently a fat-soluble vitamin derivative residue.

(item C4) the composition according to any one of the above items, wherein, in formula (1'), Y^aAnd Y^bEach independently an ester bond.

(item C5) the composition according to any one of the above items, wherein, in formula (1'), n^aAnd n^bIs 1.

(item C6) the composition according to any one of the above items, wherein, in formula (1'), R^3aAnd R^3b、Y^aAnd Y^bAnd/or X^aAnd X^bThe same is true.

(item C7-1) A composition which is a composition comprising a lipid membrane structure comprising a compound represented by the formula (1) as a constituent lipid of a membrane and a nucleic acid complex encapsulated by the lipid membrane structure,

[ solution 11]

(in the formula (1), X^aAnd X^bAre each independently X¹、X²Or X³；

[ solution 12]

s is a number of 1 or 2,

R⁴represents an alkyl group having 1 to 6 carbon atoms,

n^aand n^bAre each independently 0 or 1, respectively,

R^1aand R^1bEach independently represents an alkylene group having 1 to 6 carbon atoms,

R^2aand R^2bEach independently represents an alkylene group having 1 to 6 carbon atoms,

Y^aand Y^bEach independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond,

R^3aand R^3bIndependently represents a sterol residue, a fat-soluble vitamin derivative residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms,

the sterol residue is cholesteryl, cholestanyl, stigmasteryl, beta-sitsteryl, lanosterol or ergosteryl,

the fat-soluble vitamin is retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, vitamin D2, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol or tocotrienol),

the nucleic acid complex is a miRNA-inhibiting complex comprising RNA or an analog thereof, the miRNA-inhibiting complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the duplex structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item C7-2) the composition according to any one of the above items, which is a composition comprising a lipid membrane structure comprising a compound represented by formula (1) as a constituent lipid of a membrane, and a nucleic acid complex encapsulated by the lipid membrane structure,

[ solution 13]

(in the formula (1), X^aAnd X^bAre each independently X¹Or X²；

[ solution 14]

s is a number of 1 or 2,

R⁴represents an alkyl group having 1 to 6 carbon atoms,

n^aand n^bAre each independently a number 1 of each other,

R^1aand R^1bEach independently represents an alkylene group having 1 to 6 carbon atoms,

R^2aand R^2bEach independently represents an alkylene group having 1 to 6 carbon atoms,

Y^aand Y^bEach independently represents an ester bond, an amide bond, a urethane bond, an ether bond or a urea bond,

R^3aand R^3bIndependently represent sterol residue, fat-soluble vitamin derivative residue or aliphatic hydrocarbon group with 12-22 carbon atoms, wherein the sterol residue is cholesteryl, cholestanyl, stigmasteryl, beta-sitsteryl, lanosterol or ergosteryl,

the fat-soluble vitamin is retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, vitamin D2, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol or tocotrienol),

the nucleic acid complex is a miRNA-inhibiting complex comprising RNA or an analog thereof, the miRNA-inhibiting complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the duplex structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item C8) the composition according to any one of the above items, wherein, in the formula, X^aAnd X^bIndependently is X²。

(item C9) the composition according to any one of the above items, wherein in the formula, R^3aAnd R^3bIndependently a fat-soluble vitamin derivative residue or an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

(item C10) the composition according to any one of the above items, wherein in the formula, R^3aAnd R^3bIndependently a fat-soluble vitamin derivative residue.

(item C11) the composition according to any one of the above items, wherein the fat-soluble vitamin derivative residue is a residue derived from a reaction product of a fat-soluble vitamin having a hydroxyl group and succinic anhydride or glutaric anhydride.

(item C12) the composition of any one of the above items, wherein R^3aAnd R^3bIndependently an aliphatic hydrocarbon group having 12 to 22 carbon atoms.

(item C13) the composition according to any one of the above items, which is a composition comprising a lipid membrane structure comprising a compound represented by formula (4) as a constituent lipid of a membrane, and a nucleic acid complex encapsulated by the lipid membrane structure,

[ solution 15]

(in the formula (4), R^4aAnd R^4bEach independently an alkylene group having 8 or less carbon atoms or an oxydialkylene group,

X^1aand X^1bEach independently represents an ester bond, an amide bond, a urethane bond or an ether bond,

R^5aand R^5bEach independently represents a sterol residue, a fat-soluble vitamin residue or a carbon number13-23 aliphatic hydrocarbon group, the sterol residue is cholesteryl, cholestanyl, stigmasterol, beta-sitosteronyl, lanosteronyl or ergosterol, the fat-soluble vitamin is retinoic acid, retinol, retinal, ergosterol, 7-dehydrocholesterol, vitamin D2, cholecalciferol, dihydroergocalciferol, dihydrotachysterol, tocopherol or tocotrienol),

the nucleic acid complex is a miRNA-inhibiting complex comprising RNA or an analog thereof, the miRNA-inhibiting complex comprising at least 1 duplex structure and a miRNA-binding sequence, two strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the duplex structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item C15) the composition according to any one of the above items, wherein, in formula (4), R^4aAnd R^4bIndependently an alkylene group having 8 or less carbon atoms.

(item C16) the composition according to any one of the above items, wherein, in formula (4), X^1aAnd X^1bIs an ester bond.

(item C17) the composition according to any one of the above items, wherein, in formula (4), R^5aAnd R^5bIndependently a fat-soluble vitamin residue or an aliphatic hydrocarbon group having 13 to 23 carbon atoms.

(item C18) the composition according to any one of the above items, wherein, in formula (4), R^5aAnd R^5bIndependently a fat-soluble vitamin residue.

(item C19) the composition according to any one of the above items, wherein, in formula (4), R^5aAnd R^5bIndependently an aliphatic hydrocarbon group having 13 to 23 carbon atoms.

(item C20) the composition of any one of the above items, wherein the BNA is BNA^NC(NMe)。

(item C21) the composition according to any one of the above items, wherein the miRNA inhibition complex comprises 2 or more of the double-stranded structures, one strand each comprising a miRNA binding sequence is bound to each of two strands at one end of a first double-stranded structure of the double-stranded structures, and each of the other ends of the strands is bound to each of two strands of a second double-stranded structure of the 2 or more double-stranded structures, and is sandwiched between the 2 or more double-stranded structures.

(item C22) the composition of any one of the above items, wherein the miRNA inhibition complex comprises 2 miRNA binding sequences.

(item C23) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a structure shown below,

[ solution 16]

The structure I and II are double-stranded structures, and the structure a and b respectively contain 1 miRNA binding sequence.

(item C24) the composition of any one of the above items, wherein the miRNA binding sequence comprises 5'-CAGUGUU-3' (in the sequence, the uracil base is a thymine base if necessary) and/or 5'-CAGUAUU-3' (in the sequence, the uracil base is a thymine base if necessary).

(item C25) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises 2 miRNA binding sequences, one of the miRNA binding sequences comprises 5'-CAGUGUU-3' (in the sequence, uracil bases are thymine bases if necessary), and the other of the miRNA binding sequences comprises 5'-CAGUAUU-3' (in the sequence, uracil bases are thymine bases if necessary).

(item C26A) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a miRNA binding sequence comprising the sequence of SEQ ID NO. 1 (uracil base optionally being thymine base in the sequence), and a miRNA binding sequence comprising the sequence of SEQ ID NO. 2 (uracil base optionally being thymine base in the sequence).

(item C26B) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises a miRNA binding sequence comprising the sequence of SEQ ID NO. 3 (uracil base optionally being thymine base in the sequence) and a miRNA binding sequence comprising the sequence of SEQ ID NO. 4 (uracil base optionally being thymine base in the sequence).

(item C27A) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises the sequence of SEQ ID NO. 5 (in which the uracil base is a thymine base if necessary) and the sequence of SEQ ID NO. 6 (in which the uracil base is a thymine base if necessary).

(item C27B) the composition according to any one of the above items, wherein the miRNA-inhibiting complex comprises the sequence of SEQ ID NO. 9 (in which the uracil base is a thymine base if necessary) and the sequence of SEQ ID NO. 10 (in which the uracil base is a thymine base if necessary).

(item D1) a miRNA-inhibiting complex comprising RNA or an analog thereof for use in the treatment or prevention of a tumor, the miRNA-inhibiting complex comprising at least 1 double-stranded structure and a miRNA-binding sequence, both strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the double-stranded structure, respectively, the miRNA-inhibiting complex comprising at least 1 Bridging Nucleic Acid (BNA).

(item D2)

A miRNA-inhibitory complex for use according to any one or more of the preceding items, further having the features of any one or more of the preceding items.

(item E1) A method for preventing or treating a tumor in a subject, comprising the step of administering to the subject an effective amount of the composition, miRNA-inhibiting complex, or nucleic acid molecule of any one of the preceding items.

(item E2) the method of the above item, further having the features of any one or more of the above items.

(item F1) use of a miRNA-inhibition complex comprising RNA or an analog thereof, the miRNA-inhibition complex comprising at least 1 double-stranded structure and a miRNA-binding sequence, both strands of the miRNA-binding sequence binding to 1 of the two strands at least one end of the double-stranded structure, respectively, the miRNA-inhibition complex comprising at least 1 Bridging Nucleic Acid (BNA), for treatment or prevention of a tumor.

(item F2)

The use according to the above item, further having the features of any one or more of the above items.

It is to be noted that, for each of the above items, an invention in which two or more inventions described in each item referring to the same item are arbitrarily combined is also intended to be included in inventions described in the higher-order items referred to in them. In addition, any inventive element described in the present specification and any combination thereof is intended to be included in the present specification. In addition, the present invention is intended to include in the present invention the invention excluding any combination of elements of the dyed clothes described in the present specification or any combination thereof. In addition, when a specific embodiment is described as a preferable embodiment in the specification, for example, not only the embodiment itself but also inventions other than the embodiment from the inventions disclosed in the specification including the embodiment are disclosed.

ADVANTAGEOUS EFFECTS OF INVENTION

The miRNA inhibition activity of the improved S-TuD is enhanced compared with that of the traditional S-TuD, and the improved S-TuD is used for inhibiting miRNAs such as miR-200 family and the like, so that the prevention or treatment of tumors can be realized.

Drawings

FIG. 1 is a schematic view showing a conventional S-TuD and a partial replacement S-TuD according to the present invention.

Fig. 2 shows a typical structure of the miRNA inhibitory complex used in the present specification, and here shows a form in which 2 RNA strands containing MBS are bound to each of 2 double-stranded structures, and are sandwiched by the 2 double-stranded structures.

Fig. 3 also shows typical structures of miRNA inhibitory complexes used in the present specification, and #12 to #16 are shown here as typical examples. Here, 2 RNA strands comprising MBS were bound to the paired individual strands of the double-stranded structure, and thus the directions of the RNA strands were changed to be opposite to each other.

FIG. 4 shows the structures of (1) S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1 (with MBS against miR-141 and miR-200c) and (2) S-TuD-NCs-S10-BT6-MBSB1-S (MBS has no complementarity with miR). Lowercase letter in sequence representing substitution to BNA^NC(NMe) position.

FIG. 5 is a graph showing the change with time in body weight (g) of tumor-transplanted mice administered with (1) S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(S-TuD-141/200c) or (2) S-TuD-NCs-S10-BT6-MBSB1-S (S-TuDNCs) into the tail vein or tumor. The arrows indicate the time points at which the administration was performed.

FIG. 6 is a graph showing tumor volume (mm-TuDNCs) in tumor-transplanted mice administered with (1) S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(S-TuD-141/200c) or (2) S-TuD-NCs-S10-BT6-MBSB1-S (S-TuDNCs) into caudal vein or intratumoral tumor³) Graph of the temporal change of (2). The arrows indicate the time points at which the administration was performed.

Fig. 7 is a diagram schematically illustrating the composition of the lipid nanoparticle used in example 2.

FIG. 8 is a graph showing the change over time in body weight (g) of tumor-transplanted mice injected with S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(LNP-S-TuD 141/200c) or PBS encapsulated in lipid nanoparticles into the tail vein. The arrows indicate the time points at which the administration was performed.

FIG. 9 is a graph showing tumor volume (mm) of tumor-transplanted mice injected with S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(LNP-S-TuD 141/200c) or PBS encapsulated in lipid nanoparticles into tail vein³) Graph of the temporal change of (2). The arrows indicate the time points at which the administration was performed.

FIG. 10 shows the S-TuD structure of various miR-200 c. (41) S-TuD-200 c-1-22-pf, (42) S-TuD-200 c-1-22-pf-L18B 6, (43) S-TuD-200 c-1-22-pf-L18B 6-MBSB1 (BNA for the complementary sequence of the seed region)^NC(NMe) methylation), (44) S-TuD-200 c-1-22-pf-L18B 6-MBSB2 (BNA on the complement of the non-seed region)^NC(NMe) digestion), (45) S-TuD-200 c-1-22-pf-S10-BT 6-MBSB2 (BNA on the complement of the non-seed region)^NC(NMe) conversion).

FIG. 11 is a graph showing the change over time in body weight (g) of tumor-transplanted mice injected into the tail vein with S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(LNP-S-TuD-141/200c) or S-TuD-NCs-S10-BT6-MBSB1(LNP-S-TuD negative control) encapsulated in lipid nanoparticles. The arrows indicate the time points at which the administration was performed.

FIG. 12 is a graph showing tumor volume (mm tumor volume) of tumor-transplanted mice injected into tail vein with S-TuD-141/200 c-1-17-pf-S10-BT 6-MBSB1(LNP-S-TuD-141/200c) or S-TuD-NCs-S10-BT6-MBSB1(LNP-S-TuD negative control) encapsulated in lipid nanoparticles³) Graph of the temporal change of (2). The arrows indicate the time points at which the administration was performed.

FIG. 13 shows the structures of psiCHECK2-UT (top) and psiCHECK2-mirT (bottom).

FIG. 14 shows a schematic of a luciferase reporter vector used in the examples.

FIG. 15 shows the sequence information of psiCHECK2-T21-5p-s, psiCHECK2-T21-5p-a, psiCHECK2-T200c-3p-s, and psiCHECK2-T200c-3p-a used in the preparation of luciferase reporter vectors. These sequences are all unmodified DNA.

FIG. 16-1 shows the structure of the oligomer used.

FIG. 16-2 shows the results of reporter gene testing for miR-21 of the oligomer of FIG. 16-1. The left side shows the results at 300pM and the right side the results at 1000 pM. Error bars represent the ratio of control reporter activity to miR-21 reporter inhibitory activity. The higher the inhibitory effect of miR-21, the higher the error bar becomes.

FIG. 17-1 shows the structure of the oligomer used.

FIG. 17-2 shows the results of reporter gene testing for miR-200c for the oligomer of FIG. 17-1. The left side shows the results at 10pM and the right side shows the results at 100 pM. Error bars represent the ratio of control reporter activity to miR-200c reporter inhibitory activity. The higher the inhibitory effect of miR-200c, the higher the error bar becomes.

FIG. 17-3 shows the results of reporter gene assays performed on miR-200 c. The results obtained in H358 cells into which a TuD-141/200 c-expressing lentiviral vector was introduced are shown. Error bars represent the ratio of control reporter activity to miR-200c reporter inhibitory activity. The higher the inhibitory effect of miR-200c, the higher the error bar becomes.

Detailed Description

The present invention will be explained below. Throughout the specification, the singular form of expressions should be understood to include the plural form of the concept when not specifically stated. Thus, articles in the singular (e.g., "a," "an," "the," etc. in english) should be understood to also encompass the concept of their plural forms when not specifically stated. In addition, unless otherwise specified, terms used in the present specification should be understood to be used in a meaning commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification, including definitions, will control.

< miRNA inhibitory Complex >

In the present invention, it has been found that the miRNA inhibitory complex described in detail below can be used as a drug for the prevention or treatment of various diseases, and in particular, tumor treatment or prevention can be performed. In some embodiments of the invention, compositions comprising miRNA-inhibitory complexes described in detail below are provided for the treatment or prevention of tumors.

The invention further provides compositions comprising the miRNA-inhibitory complexes detailed below and a vector for nucleic acid delivery. By combining with such a vector for nucleic acid delivery, the miRNA inhibitory complex described in the present specification can be better delivered to a target.

For details of the improved miRNA inhibitory complexes used in the present specification, PCT/JP2016/004252, which is incorporated herein by reference, can efficiently and specifically inhibit mirnas. The miRNA inhibitory complex used in the present specification is characterized by comprising at least 1 double-stranded structure, at least 1 strand comprising a MiRNA Binding Sequence (MBS) bound to both strands of at least one end of the double-stranded structure, comprising at least 1 Bridging Nucleic Acid (BNA). The inhibitory complex used in the present invention is sometimes referred to as "S-TuD". It should be noted that the present invention is also applicable to the present inventionThis double stranded structure is referred to as the "first" double stranded structure in order to be distinguishable from other double stranded structures that may be included in the complexes used in this specification. The complex used in the present specification may be a single strand (i.e., 1 molecule covalently bonded) or may be composed of, for example, a single strand, a double strand, or two or more strands. For example, as long as the complex comprising double-stranded RNA comprises at least 1 bridging nucleic acid in the complex (BNA, e.g., BNA)^NC(NMe)) is included in the scope of the complexes used in the present invention, wherein the double-stranded RNA is RNA to which 1 MBS-containing RNA strand is bound on each of both strands at one end of a double-stranded structure. In addition, for example, one RNA strand comprising at least 1 MBS may be bound to both strands at one end of the double-stranded structure. In this case, the two strands at one end of the double-stranded structure are linked by the RNA strand comprising MBS. The RNA connecting both strands of the double-stranded structure comprises at least 1 MBS and may comprise, for example, 2,3 or more than 3. The double-stranded structure comprises a stem-loop structure or a hairpin. That is, the double-stranded structure may be a double-stranded structure contained in a stem-loop structure or a hairpin. The improved miRNA inhibitory complex is expected to have an improved in vivo tumor inhibitory effect due to its high inhibitory efficiency and high serum stability.

In the present invention, the "non-seed" region means: bases other than bases at positions 2 to 8 from the 5 'end of miRNA required for miRNA activity in the miRNA sequence, specifically bases at positions 9 to 21 from the 5' end of miRNA. In the complex used in the present invention, the "non-seed binding region" means: sequences within MBS that bind to non-seed regions of mirnas with high complementarity, "stem region" refers to double-stranded structures. The BNAs contained may or may not be contained in the non-seed binding region, or may not be contained in the stem region (see fig. 1). In the case where BNA is contained only in the non-seed binding region or BNA is contained only in the stem region, it is understood that the activity is similarly enhanced depending on the contained BNA.

The miRNA inhibitory complex used in the present invention may be a structure having a double-stranded structure comprising at least 1 RNA or an analog thereof. The complex preferably comprises one or two molecules of a molecule comprising RNA or an analog thereof.

In the present specification, "miRNA-binding sequence (MBS)" means: a sequence that binds to a miRNA. MBS comprises at least a portion complementary to miRNA so that it can bind to miRNA. As shown in japanese patent No. 4936343, MBS may or may not be a sequence completely complementary to miRNA. For example, MBS may be a native RNA sequence that targets miRNA. MBS comprises, for example, complementary bases of at least 10 bases, for example, 11 bases or more, 12 bases or more, 13 bases or more, 14 bases or more, 15 bases or more, 16 bases or more, 17 bases or more, 18 bases or more, 19 bases or more, 20 bases or more, 21 bases or more, 22 bases or more, 23 bases or more, or 24 bases or more, continuously or discontinuously with respect to miRNA. The complementary bases may be contiguous or may have 3 or fewer, 2 or fewer, preferably 1 notch. Gaps can be MBS-and/or miRNA-sided unpaired (bulges), and for 1 gap, there can be only bulge bases on one strand, or unpaired bases on both strands. For example, it can be designed to include unpaired bases at least on the MBS side. Regarding the number of bases of the bulge and the mismatch, the number of bases of the bulge and the mismatch is, for example, 6 bases or less, preferably 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less, or 1 base per strand. The MBS that can form a bulge sometimes shows a higher miRNA inhibitory effect than the MBS formed from a completely complementary sequence. Therefore, to obtain a higher miRNA inhibitory effect, MBS can be designed in a convex manner. For example, the MBS in which the 10 th and/or 11 th bases from the 3 'end of the MBS are not complementary to the miRNA or contain an extra base between the 10 th and 11 th positions (or the MBS in which the 10 th and/or 11 th bases from the 5' end of the target sequence (sequence hybridizing with the MBS) in the miRNA are non-complementary bases to the MBS or contain an unpaired base between the 10 th and 11 th nucleotides) is not easily decomposed, and a high activity can be expected. However, when a modified base having high resistance to degradation is used, it is not necessary to include a bulge. In this case, for example, the MBS may be designed so that the 10 th and 11 th bases from the 5' end of the miRNA are not paired, or, for example, the 9 th to 11 th, 10 th to 12 th or 9 th to 12 th bases are not paired. In addition, a mode in which there is no unpaired base on the miRNA side, but there is an unpaired base between the 10 th and 11 th positions from the 3 'end (or between the 10 th and 11 th positions from the 5' end of the target sequence (sequence hybridizing with MBS) in miRNA) on the MBS side is also permissible. Unpaired bases may be present on the miRNA side and/or the MBS side, preferably at least on the MBS side. The number of unpaired nucleotides in each strand can be suitably adjusted, for example, to 1 to 6 nucleotides, preferably 1 to 5 nucleotides, more preferably 3 to 5, e.g., 3, 4 or 5 nucleotides. In addition, it is known that for the identification of miRNA targets, it is important to match the bases (seed regions) at positions 2-8 from the 5' end of miRNA (Jackson ALet al, RNA12(7): 1179-. Actually, miRNA-inhibitory RNA used in the present specification can effectively inhibit miRNA even if it is RNA having MBS that matches only seed regions and has only low complementarity with other regions. The MBS in the present invention is preferably an MBS that is completely complementary to a seed region of a miRNA (base at positions 2 to 8 from the 5' end of the miRNA). In this case, the G: U pair (U: G pair) can also be regarded as complementary, preferably only G: C (C: G) and A: U (U: A) are regarded as complementary. Further, as the MBS in the present invention, it is preferable that the MBS is completely complementary to a seed region of the miRNA (base at positions 2 to 8 from the 5' end of the miRNA) and includes complementary bases of at least 8 bases, more preferably 9 bases, and still more preferably 10 bases continuously with respect to the miRNA. Further, the MBS in the present invention preferably includes complementary bases of 11 bases or more, more preferably 12 bases or more, and still more preferably 13 bases or more in total to the miRNA.

MBS is preferably a sequence that hybridizes to a miRNA sequence under physiological conditions. Under physiological conditions, the following means: for example, 150mM NaCl, 15mM sodium citrate, pH7.0, 37 ℃. More preferably, MBS is a sequence that hybridizes to a miRNA sequence under stringent conditions. Stringent conditions refer to: for example, 1 XSSC (1 XSSC is 150mM NaCl, 15mM sodium citrate, pH7.0) or 0.5 XSSC at 42 ℃, more preferably 1 XSSC or 0.5 XSSC at 45 ℃, and still more preferably 1 XSSC or 0.5 XSSC at 50 ℃. In hybridization, for example, one of the RNA including the miRNA sequence and the RNA including MBS is labeled, and the other is immobilized on a membrane to hybridize the two. As for the conditions for hybridization, for example, it can be carried out in a solution containing 5XSSC, 7% (W/V) SDS, 100. mu.g/ml denatured salmon sperm DNA, 5 XDenhardt's solution (1 XDenhardt's solution containing 0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% polysome (Ficoll)) and at 37 ℃ or 45 ℃ or 50 ℃ for example. After incubation for a sufficient period of time (e.g., 3, 4,5, or 6 hours or more), washing is performed under the conditions described above to detect whether hybridization of the labeled nucleic acid has occurred, thereby allowing determination of whether the nucleic acid hybridizes under the conditions.

Alternatively, MBS preferably shows high homology to the complement of the miRNA sequence. High homology means that: for example, a nucleotide sequence having 70% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity. The identity of the base sequences can be determined, for example, using the BLAST program (Altschul, S.F.et al., J.Mol.biol.215: 403-. For example, a search can be performed in the BLAST webpage of the NCBI (National Center for Biotechnology Information, U.S. National Center for Biotechnology Information) using default parameters (Altschul S.F.et al, Nature Gene.3: 266-. For example, the blast2sequences program (Tatiana A et al, FEMS Microbiol Lett.174:247-250,1999), which compares 2sequences, can be used to establish an alignment of two sequences to determine sequence identity. The outer gaps of the base sequences of the miRNA sequences are negligible, and the inner gaps are treated in the same manner as the mismatches, for example, to calculate the identity of the miRNA sequences in the alignment with respect to the entire base sequences (total base length obtained by adding the gaps entering the inner sides of the sequences). However, as shown in Japanese patent No. 4936343, the mismatch between MBS and miRNA may increase the inhibitory activity of miRNA. Thus, for example, gaps in the inserted miRNA sequences located in the inner side of the alignment are preferably ignored for calculating identity.

Alternatively, the MBS may be composed of a sequence obtained by insertion, substitution, and/or deletion of 1 or several bases with respect to the complementary sequence of the miRNA sequence. The MBS may be a sequence having an insertion, substitution, and/or deletion of 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less or 1 base with respect to the complementary sequence of the miRNA sequence. Alternatively, the MBS may be a sequence having an insertion of 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, 2 bases or less or 1 base relative to the complementary sequence of the miRNA sequence. When MBS is a sequence with a mismatch, the inhibitory activity of miRNA is sometimes higher compared to a sequence perfectly complementary to the miRNA sequence. It is believed that this is due to the reduced expression level of miRNA-inhibitory RNA when MBS is a fully complementary sequence, cleaved by RISC containing miRNA. However, when a modified base having high resistance to degradation is used, it is not necessary to include a bulge. In particular, high activity can be expected for MBS designed in such a manner that the 10 th and/or 11 th bases from the 3 'end of the MBS do not pair when the MBS is hybridized with miRNA (or the 10 th and/or 11 th bases from the 5' end of the target sequence on the side of miRNA hybridized with the MBS do not pair when hybridized with the MBS) or that unpaired bases are included between the 10 th and 11 th nucleotides. Such unpaired bulges can be, for example, MBS-side bulges, with the bulge-forming bases being 1 to 6 bases, preferably 1 to 5 bases, and more preferably 3 to 5 bases (e.g., 3, 4, or 5 bases). MBS may be composed of RNA, or may comprise or consist of nucleic acid analogs. In particular, by using a nucleic acid analog as a cleavage site of MBS (e.g., 10 th and/or 11 th base from the 3' end of MBS), the occurrence of cleavage can be avoided, and an improvement in miRNA inhibitory effect can be expected. In addition, it is also suitable to use Nucleic Acids having a backbone such as phosphorothioate, 2' -O-methyl or the like, or a sugar (Krutzfeldt, J.et al, Nucleic Acids Res.35: 2885-.

In the present specification, the miRNA targeted by the miRNA inhibitory complex used is not particularly limited. As long as it has a miRNA structure, it can be applied to miRNA derived from any species such as plants, nematodes, and vertebrates. It is known that the sequence of miRNA is very high in a large number of organisms including human, mouse, chicken, zebrafish and Arabidopsis thaliana (refer to miRBase:: sequence: microrna.sanger.ac.uk/Sequences /). For example, mirnas can be targeted to mammals including mice, rats, goats, etc., primates including monkeys, and humans. Examples thereof include miRNAs of miR-200 family (for example, miR-200a, miR-200b, miR-200c, miR-141 and miR-429), and preferably include miR-200c and miR-141. Further, examples of the miRNA that can be used as a target include miRNA of miR-21 and miR-17-92 cluster (e.g., miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, miR-92a-1), miR-155, miR-133a, miR-196b, miR-197, miR-205, miR-125b, miR-135b, miR-106a, miR-10a/10b, miR-146a, miR-182, miR-96 and the like.

In one embodiment, the miRNA inhibition complex used in the present specification includes a second double-stranded structure in addition to a first double-stranded structure, and has a structure in which one RNA strand including MBS is bound to each of two strands located at one end of the first double-stranded structure, and each other end of the RNA strand is bound to each of two strands located at one end of the second double-stranded structure, so that the RNA strand is sandwiched between the first double-stranded structure and the second double-stranded structure. For example, a miRNA inhibitory complex comprises a plurality of miRNA binding sequences, e.g., 2 to 5 miRNA binding sequences. The way in which the miRNA inhibitory complex comprises a plurality of miRNA binding sequences is advantageous in inhibiting a plurality of mirnas simultaneously, and is useful in treatment or prevention of tumors that are effectively treated or prevented by inhibiting a plurality of mirnas. In one embodiment, the miRNA inhibition complex comprises 2 miRNA binding sequences. The double-stranded structure may be two strands, or may be 4 strands such as G-quadruplex (G-quadruplex). For example, in one embodiment of the present invention, the double-stranded structure further includes a second double-stranded structure in addition to the first double-stranded structure, and the two strands forming the ends of the first double-stranded structure to which MBS binds are bound are each bound with a structure in which one RNA strand including MBS is bound, and the other ends of the RNA strands are each bound with the two strands of the second double-stranded structure, so that the RNA strands are sandwiched between the first double-stranded structure and the second double-stranded structure. The RNA complex has, for example, the following structure: has at least 2 double-stranded structures, and 4 RNA strands constituting the 2 double-stranded structures are directly bound to MBS-containing RNA without intervening any of the remaining 3 strands. If such miRNA inhibitory complexes are more clearly described, they are miRNA inhibitory complexes in which 2 RNA strands containing MBS are bound to each of 2 double-stranded structures so as to be sandwiched between the 2 double-stranded structures (fig. 2). That is, the present invention includes RNA in which RNA strands a and b are sandwiched by double-stranded structures I and II and which includes 1 or more MBS in a and b, respectively, as an RNA complex having the structure of fig. 2. 2 RNA strands comprising MBS were bound to each paired strand of the double-stranded structure, and thus the directions of the RNA strands were reversed with respect to each other (FIGS. 3, #12 to # 16). By adding MBS to each strand of the double strand in this manner, higher miRNA inhibitory activity can be exhibited.

The 2 RNA strands containing MBS, which are present so as to be sandwiched by 2 double-stranded structures, each contain 1 or more MBS. The sequences of these MBS's may be the same or different. In addition, the miRNA may be targeted to the same sequence, or may be bound to a different target miRNA. For example, more than 2, e.g., 2,3, 4 or 5 MBS's may be included in 1 chain (fig. 3, #12 to # 16). For example, 1 or 2 MBS's may be contained in each strand sandwiched by 2 double-stranded structures. For example, a miRNA inhibitory complex used in the present specification may comprise a total of 2 MBS's, which 2 MBS's may be the same sequence or sequences binding to the same miRNA, or may be different sequences or sequences binding to different mirnas.

In the present specification, each strand of the miRNA inhibitory complex used in the present specification, which is a double-stranded paired strand, is usually an RNA molecule as described above, and one or both ends of the double strand may be linked to form a straight chain or a loop. The term "linear" merely means having a terminal with respect to a cyclic term, and does not mean that a secondary structure is not formed. The miRNA inhibitory complex composed of a linear single-stranded RNA can be prepared, for example, by one-time RNA synthesis. For example, in the case of containing 2 double-stranded structures, two strands at one end of the second double-stranded structure (the side not binding MBS) may be connected with a loop, so that a single strand is formed as a whole. In the sequence for connecting double strands, 1 or more MBS may be included (e.g., fig. 3, #13, #14, # 16). To make the sequence as compact as possible, the duplexes may be joined with short loops. For example, the double strands can be ligated by using a sequence of 1 to 10 bases, preferably 1 to 8 bases, 2 to 6 bases, 3 to 5 bases, for example, 4 bases. The sequence is not particularly limited. Examples thereof include 5 '-GUCA-3'. For example, the present invention includes RNA having the structure of #13 in FIG. 3, wherein RNA strands a and b are sandwiched by double-stranded structures I and II, and the double-stranded structure II forms a hairpin (or stem-loop), and wherein 1 or more MBS is contained in each of a and b.

In the present specification, the sequence of the double-stranded structure included in the miRNA inhibitory complex to be used is not particularly limited, and may be any base length. With respect to the preferred embodiments, additional details will be provided below.

The sequence of base pairs forming a double-stranded structure can be appropriately designed so as to form a double strand specifically and stably in the miRNA inhibitory complex. For example, it is preferable to avoid a homopolymeric sequence in which the same base is continuous over a long period (for example, 8 bases or more, preferably 7 bases or more, more preferably 5 bases or more, more preferably 4 bases or more, and more preferably 3 bases or more). In addition, it is preferable to avoid a sequence in which a sequence of several bases is repeated in tandem such as a two-base repeat sequence and a3 to 4-base repeat sequence. The GC content of the double-stranded portion may be suitably adjusted, and is, for example, 12% to 85%, preferably 15% to 80%, 20% to 75%, 25% to 73%, 32% to 72%, 35% to 70%, 37% to 68%, or 40% to 65%. Examples of such sequences include, but are not limited to, the sequences of stem I and stem II shown in Japanese patent No. 4936343. The 4-strand may be a G-quadruplex, and specifically may be a sequence GGG-loop-GGG-loop-GGG-loop-GGG. The sequence of the loop is appropriately selected, and for example, the 3 loops may be 1 base (for example, M (a or C)) or 3 bases.

The MBS and the double-stranded structure can be directly connected or connected by other sequences. For example, MBS can be bound to the ends of the double stranded structure by means of a suitable linker or spacer sequence. Even if MBS is directly linked to a double-stranded portion, significant inhibitory activity can be obtained, but the inhibitory effect on miRNA will be further increased by adding a linker (also referred to as a spacer). The linker or spacer sequence between the MBS sequence and the double stranded structure may increase the accessibility of MBS to mirnas present in RISC. The length of the linker or spacer sequence may be appropriately adjusted, and is, for example, 1 to 10 bases, preferably 1 to 9 bases, 1 to 8 bases, 1 to 7 bases, 1 to 6 bases, 1 to 5 bases, 1 to 4 bases or 1 to 3 bases. For example, in the case of connecting more than 2 MBS, the connection may be made by means of a linker or a spacer sequence. The sequence of the linker or spacer is not particularly limited, and may be, for example, a sequence consisting of a and/or C or a sequence containing a larger amount of a and/or C than other bases. In addition, care is preferably taken to avoid the formation of stable base pairs between the linker or spacer and the facing linker or spacer or MBS. For example, AGA, AAC, CAA, ACC, CCA, or a sequence including any of these may be used. A pair of splice or spacer sequences added on both sides of the MBS may be set as a reverse sequence (mirror sequence). For example, AAC may be added to the 5 'side of MBS and CAA may be added to the 3' side.

In the present specification, the nucleic acid constituting the miRNA inhibitory complex used is characterized by being modified with the specific modified nucleic acid of the present invention, but may contain modified nucleic acids other than the specific modified nucleic acid. For example, the nucleotides that make up a nucleic acid may comprise natural nucleotides, modified nucleotides, artificial nucleotides, or combinations thereof, in addition to the particular modified nucleic acids of the invention. In addition, as long as the nucleic acid contained in the miRNA inhibitory complex used in the present specification includes the specific modified nucleic acid mentioned in the present specification, the nucleic acid other than the specific modified nucleic acid may be composed of RNA, may be an RNA-DNA chimera, may be another nucleic acid analog, and may include any combination thereof. The nucleic acid includes not only a nucleic acid bonded via a phosphodiester bond but also a nucleic acid having an amide bond or other skeleton (e.g., Peptide Nucleic Acid (PNA)) as long as the nucleic acid includes the specific modified nucleic acid of the present invention. Nucleic acid analogs include, for example, natural and artificial nucleic acids, which can be nucleic acid derivatives, nucleic acid analogs, nucleic acid derivatives, and the like. Such nucleic acid analogs are well known in the art and include, for example, phosphorothioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2 "-O-methyl ribonucleotide, Peptide Nucleic Acid (PNA), but are not limited thereto. As the PNA skeleton, a skeleton containing aminoethylglycine, polyamide, polyethyl, polythioamide, polysulfinil, polysulfonamide or a combination thereof (Krutzfeldt, J.et al, Nucleic Acids sRs.35: 2885-2892; Davis, S.et al, 2006, Nucleic Acids Res.34: 2294-2304; Boutra, A.et al, 2003), Nucleic Acids Res.31: 4973-4980; hutvagner, G.et al, 2004, PLoBiol.2: E98; chan, J.A.et al, 2005, Cancer Res.65: 6029-; esu, C.et al, 2004, J.biol.chem.279: 52361-52365; esu, C.et al, 2006, Cell Metab.3: 87-98).

(bridging nucleic acid (BNA) used in the present invention)

The miRNA inhibitory complex used in the present invention is characterized by containing a stabilized nucleic acid, i.e., a modified nucleic acid that promotes double strand formation as a specific modified nucleic acid, and is characterized by containing a Bridged Nucleic Acid (BNA) in a broad sense, for example.

In the present specification, "Bridged Nucleic Acid (BNA)" (BNA refers to both bicylic Nucleic Acid and Bridged Nucleic Acid; also referred to as "Bridged Nucleic Acid", "Bicyclic Nucleic Acid", or "bridge/Bicyclic Nucleic Acid") refers to: an arbitrary modified nucleic acid in which the 2 '-position and the 4' -position of the nucleic acid are linked (bridged) so that the loop structure is 2 (bicyclic).

In 1 exemplary embodiment, as the stabilized nucleic acid (i.e., a modified nucleic acid that promotes double strand formation) used in the present invention, a bridging nucleic acid may be used. For example, japanese patent No. 4731324, praadep s. pallan et al, Chem commu (Camb).2012August 25; 48(66), 8195-8197.doi:10.1039/C2cc32286b, and examples thereof include Locked Nucleic Acids (LNA), ethylene nucleic acids such as 2 '-O, 4' -C-ethylene bridged nucleic acids (ENA)), other Bridged Nucleic Acids (BNA), Hexitol Nucleic Acids (HNA), morpholino nucleic acids, tricyclo-DNA (tcDNA), polyether nucleic acids (see, for example, U.S. Pat. No. 5,908,845), cyclohexene nucleic acids (CeNA), and combinations thereof.

In the present specification, "substituted" means: a specific hydrogen atom in an organic compound such as a Bridged Nucleic Acid (BNA) is substituted with another atom or group of atoms.

In the present specification, the "substituent" means: an atom or a functional group substituting for another atom or functional group in the chemical structure of a Bridged Nucleic Acid (BNA) or the like.

In the present specification, examples of the substituent that can be used in the miRNA inhibitory complex to be used include, but are not limited to, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, carbocyclyl, heterocyclyl, halogen, hydroxyl, thiol, cyano, nitro, amino, carboxyl, carbamoyl, acyl, acylamino, thiocarboxyl, amide, substituted carbonyl, substituted thiocarbonyl, substituted sulfonyl and substituted sulfinyl. The substituent may have a substituent other than hydrogen.

In the present specification, unless otherwise specified, substitution means: 1 or more than 2 hydrogen atoms in an organic compound or substituent are substituted with other atoms or atomic groups or form double bonds or triple bonds. It is also possible to remove 1 hydrogen atom and substitute a substituent having a valence of 1 or form a double bond together with a single bond, and it is also possible to remove 2 hydrogen atoms and substitute a substituent having a valence of 2 or form a triple bond together with a single bond.

In the present specification, "alkyl" means: the 1-valent group formed by removing one hydrogen atom from an aliphatic hydrocarbon (alkane) such as methane, ethane or propane, is usually C_nH_2n+1-represents (here, n is a positive integer). The alkyl group may be straight or branched. Specific examples thereof include a C1-C2 alkyl group, a C1-C3 alkyl group, a C1-C4 alkyl group, a C1-C5 alkyl group, a C1-C6 alkyl group, a C1-C7 alkyl group, a C7-C7 substituted alkyl group, a C7-C7 alkyl group, and a C7-C7 substituted alkyl group. Here, for example, the C1-C10 alkyl group means: a linear or branched alkyl group having 1 to 10 carbon atoms. In the present specification, "substituted alkyl" means: an alkyl group in which H in the alkyl group is substituted with a substituent defined in the present specification. Specifically, although not limited thereto, CH may be mentioned₃OCH₂-、CH₃OCH₂CH₂-、CH₃OCH₂CH₂CH₂-、HOCH₂-、HOCH₂CH₂-、HOCH₂CH₂CH₂-、NCCH₂-、NCCH₂CH₂-、NCCH₂CH₂CH₂-、FCH₂-、FCH₂CH₂-、FCH₂CH₂CH₂-、H₂NCH₂-、H₂NCH₂CH₂-、H₂NCH₂CH₂CH₂-、HOOCCH₂-、HOOCCH₂CH₂-、HOOCCH₂CH₂CH₂-。

In the present specification, "alkylene" means: the 2-valent group formed by losing two hydrogen atoms from an aliphatic hydrocarbon (alkane) such as methane, ethane, propane, etc., usually represented by-C_nH_2n-represents (here, n is a positive integer). The alkylene group may be straight or branched. In the present specification, "substituted alkylene" means: an alkylene group in which H of the alkylene group is substituted with the above-mentioned substituent. Specific examples thereof include C1 alkylene, C1-C2 alkylene, C1-C3 alkylene, C1-C4 alkylene, C1-C5 alkylene, and C1-C6 alkyleneC1-C7 alkylene, C1-C8 alkylene, C1-C9 alkylene, C1-C10 alkylene, C1-C11 alkylene, C1-C20 alkylene, C1-C2 substituted alkylene, C1-C3 substituted alkylene, C1-C4 substituted alkylene, C1-C5 substituted alkylene, C1-C6 substituted alkylene, C1-C7 substituted alkylene, C1-C8 substituted alkylene, C1-C9 substituted alkylene, C1-C10 substituted alkylene, C1-C11 substituted alkylene, or C1-C20 substituted alkylene. Here, for example, C1-C10 alkylene means: a linear or branched alkylene group having 1 to 10 carbon atoms. Further, for example, a C1-C10 substituted alkylene group means: a C1-C10 alkylene group in which 1 or more hydrogen atoms are substituted with a substituent. In the present specification, the "alkylene group" may contain 1 or more atoms selected from an oxygen atom and a sulfur atom.

In the present specification, "cycloalkyl" means: an alkyl group having a cyclic structure. "substituted cycloalkyl" refers to: cycloalkyl wherein H of the cycloalkyl is substituted by the above-mentioned substituent. Specific examples thereof include C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalkyl, C3-C8 cycloalkyl, C3-C9 cycloalkyl, C3-C10 cycloalkyl, C3-C11 cycloalkyl, C3-C20 cycloalkyl, C3-C4 substituted cycloalkyl, C3-C5 substituted cycloalkyl, C3-C6 substituted cycloalkyl, C3-C7 substituted cycloalkyl, C3-C8 substituted cycloalkyl, C3-C9 substituted cycloalkyl, C3-C10 substituted cycloalkyl, C3-C11 substituted cycloalkyl, and C3-C20 substituted cycloalkyl.

In the present specification, "alkenyl group" means: 1-valent group obtained by losing one hydrogen atom from aliphatic hydrocarbon having one double bond in the molecule, usually C_nH_2n-1-represents (here, n is a positive integer of 2 or more). "substituted alkenyl" means: an alkenyl group in which H of the alkenyl group is substituted with the above-mentioned substituent. Specific examples thereof include C2 to C3 alkenyl groups, C2 to C4 alkenyl groups, C2 to C5 alkenyl groups, C2 to C6 alkenyl groups, C2 to C7 alkenyl groups, C2 to C8 alkenyl groups, C2 to C9 alkenyl groups, C2 to C10 alkenyl groups, C2 to C11 alkenyl groups or C2 to C20 alkenyl groups, C2 to C3 substituted alkenyl groups, C2 to C4 substituted alkenyl groups, C4 to C4 substituted alkenyl groups, and C4 to C4 substituted alkenyl groupsOr C2-C20 substituted alkenyl. Here, for example, the C2-C10 alkyl group means a straight-chain or branched alkenyl group having 2 to 10 carbon atoms. Further, for example, the C2 to C10 substituted alkenyl group means a C2 to C10 alkenyl group in which 1 or more hydrogen atoms are substituted with a substituent.

In the present specification, "aryl" means: 1 hydrogen atom bonded to the ring of an aromatic hydrocarbon is removed, and in this specification, an aryl group is included in a carbocyclic group. Derived from benzene is phenyl (C)₆H₅-) derived from toluene is tolyl (CH)₃C₆H₄-) and derived from xylene is xylyl ((CH)₃)₂C₆H₃-, derived from naphthalene is naphthyl (C)₁₀H₈-)。

In the present specification, "aralkyl" means: an alkyl group in which 1 hydrogen atom of the alkyl group is substituted with an aryl group. Specific examples of the aralkyl group include benzyl, phenethyl (phenylethyl), 1-naphthylethyl and the like.

In the present specification, "acyl" means: a 1-valent group formed by removing OH from a carboxylic acid. As a typical example of the acyl group, acetyl (CH) may be mentioned₃CO-), benzoyl (C)₆H₅CO-) and the like. "substituted acyl" refers to: a group obtained by substituting the hydrogen of an acyl group with the above substituent.

In the present specification, "sulfonyl group" is a group which is a group containing a characteristic group-SO₂General term for the group of (A-E). "substituted sulfonyl" means: a sulfonyl group substituted with the above-mentioned substituent.

In the present specification, "silyl group" means SiR which is generally used₁R₂R₃A group represented by (herein, R)₁、R₂、R₃Each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, alkoxy, carbocyclyl, heterocyclyl). Specific examples of these include trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, triisopropylsilyl and t-butyldiphenylsilyl.

In the present specification, the "functional molecular unit substituent" means: a group comprising a labeling molecule (including, for example, a fluorescent molecule, a chemiluminescent molecule, a molecular species including a radioisotope, etc.), a DNA or RNA cleavage active molecule, an intracellular or nuclear migration signal peptide, etc.

In one embodiment, the BNA may be BNA bridged via at least 1 atom selected from the group consisting of oxygen and carbon on the 2 'side, via at least 1 atom selected from the group consisting of carbon and nitrogen on the 4' side.

In representative embodiments, the BNA used in the invention is a2 ', 4' substituted bridging nucleic acid as represented by BNA-1 below,

[ solution 17]

(in the formula (BNA-1), R₁、R_1’、R₂、R_2’And R₃Each independently represents a group selected from the group consisting of a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted acyl group, a substituted or unsubstituted sulfonyl group, a substituted or unsubstituted silyl group, and a functional molecular unit substituent, for example, although not limited thereto, there may be mentioned substituted or unsubstituted phenoxyacetyl, alkyl group having 1 to 5 carbon atoms, alkenyl group having 1 to 5 carbon atoms, aryl group having 6 to 14 carbon atoms, methyl group substituted with 1 to 3 aryl groups, lower aliphatic or aromatic sulfonyl group such as methanesulfonyl group or p-toluenesulfonyl group, or aromatic acyl group having 1 to 5 carbon atoms such as acetyl group, benzoyl group or the like, n is an integer of 1 to 3, and q is an integer of 0 or 1. ).

Base is purin-9-yl, 2-oxo-pyrimidin-1-yl or derivatives thereof, and examples thereof include, but are not limited to, the representative 6-aminopurin-9-yl (i.e., adenine), 2-amino-6-chloropurin-9-yl, 2-amino-6-fluoropurin-9-yl, 2-amino-6-bromopurin-9-yl, 2-amino-6-hydroxypurin-9-yl (i.e., guanidino), 6-amino-2-chloropurin-9-yl, 6-amino-2-fluoropurin-9-yl, 2, 6-dimethoxypurin-9-yl, 2-oxo-pyrimidin-1-yl groups of the present invention exemplified in Japanese patent No. 4731324, 2, 6-dichloropurin-9-yl, 6-mercaptopurine-9-yl, 2-oxo-4-amino-1, 2-dihydropyrimidin-1-yl (i.e., cytosine), 2-oxo-4-amino-5-fluoro-1, 2-dihydropyrimidin-1-yl, 4-amino-2-oxo-5-chloro-1, 2-dihydropyrimidin-1-yl, 2-oxo-4-methoxy-1, 2-dihydropyrimidin-1-yl, 2-oxo-4-mercapto-1, 2-dihydropyrimidin-1-yl, 2-oxo-4-hydroxy-1, 2-dihydropyrimidin-1-yl (i.e., uracil), 2-oxo-4-hydroxy-5-methyl-1, 2-dihydropyrimidin-1-yl (i.e., thymidylyl), 4-amino-5-methyl-2-oxo-1, 2-dihydropyrimidin-1-yl (i.e., 5-methylcytosinyl), 9- β -D-ribofuranosylhypoxanthine (i.e., inosinyl) and their derivatives, adenine, thymidyl, guanidino, uracil, inosine, cytosinyl, and 5-methylcytosine groups and their derivatives can be preferably exemplified.

In another representative embodiment, the BNA used in the invention comprises the 2 ', 4' substituted bridging nucleic acid as shown in BNA-2 below,

[ solution 18]

(in the formula (BNA-2), R₃The substituent is selected from the group consisting of a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted acyl group, a substituted or unsubstituted sulfonyl group, a substituted or unsubstituted silyl group, and a functional molecular unit substituent, and examples thereof include, but are not limited to, phenoxyacetyl, a C1-5 alkyl group, a C1-5 alkenyl group, a C6-14 aryl group, a C1-3 aryl-substituted methyl group, a lower aliphatic or aromatic sulfonyl group such as a methanesulfonyl group or p-toluenesulfonyl group, an aliphatic acyl group such as an acetyl group having a C1-5 carbon number, and an aromatic acyl group such as a benzoyl group, m is an integer of 0 to 2, and n is an integer of 1 to 3. ). Base is the same as that described in BNA-1,adenine, guanidino, thymidylyl, uracil, inosine, cytosine, and 5-methylcytosine, and their derivatives can be preferable.

In another representative embodiment, the BNA used in the invention is the following BNA-3:

[ solution 19]

(in the formula (BNA-3), R₂And R_2’Each independently represents a group selected from the group consisting of a hydrogen atom, a substituted or unsubstituted alkyl group, a substituted or unsubstituted alkenyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted aralkyl group, a substituted or unsubstituted acyl group, a substituted or unsubstituted sulfonyl group, a substituted or unsubstituted silyl group, and a functional molecular unit substituent, for example, although not limited thereto, a methyl group, an O-methoxyethyl group, the same Base as that described in BNA-1, and preferably an adenine group, a guanidinium group, a thymine group, a uracil group, a sarcosine group, a cytosine group, a 5-methylcytosine group, and derivatives thereof may be cited. Wherein n is an integer of 1 to 3, R₂Or R_2’Are not all hydrogen. )

The BNA having a branch in the bridged chain is not limited thereto, but examples thereof include BNA (cEt),

[ solution 20]

(cEt: 2 ', 4' -Limited Ethyl). Bna (cet) is known to have thermal stability and mismatch discrimination similar to those of conventional LNAs, but to have improved nuclease stability.

In representative embodiments, the BNA used in the present invention may be:

[ solution 21]

(in the present specification, unless otherwise specified, "BNA" means^NC(NMe) ", sometimes also denoted" (2 ', 4' -) BNA^NC". ) Here, Base is defined as the same as above, and is preferably selected from the group consisting of adenine group, thymidyl group, guanidino group, uracil group, inosine group, cytosine group and 5-methylcytosine group.

In the present specification, "protecting group" means: groups used to protect functional groups in a particular chemical reaction. In the present specification, a protecting group may be referred to as "PG".

Preferably, BNA is used^NC(NMe) or LNA as BNA, more preferably BNA^NC(NMe) as BNA.