Double-stranded RNA containing carbamoylethyl modification

Abstract

The purpose of the present invention is to provide a chemically modified siRNA having further enhanced metabolic stability while maintaining activity.　The present invention provides a double-stranded RNA capable of inhibiting expression of a target RNA, wherein the double-stranded RNA contains a sense strand and an antisense strand, the antisense strand has 14-40 nucleotides and has sufficient complementarity with the target RNA to mediate RNA interference, the sense strand has 14-40 nucleotides and has complementarity with the antisense strand, and the double-stranded RNA contains at least one 2'-O-XCE nucleotide.

Description

Double-stranded RNA containing carbamoylethyl modifications

[0001] The present invention relates to a double-stranded RNA containing a carbamoylethyl modification (2'-O-XCE nucleotide) capable of inhibiting the expression of a target RNA, a pharmaceutical composition containing the same, and a method for regulating the expression of a target RNA using the same.

[0002] One method for inhibiting the expression of a target gene in a cell, tissue, or individual involves introducing double-stranded RNA (SIDR) called small interfering RNA (siRNA) into the cell, tissue, or individual. The introduction of SIDR results in the degradation of target RNAs with homology to the SIDR sequence, thereby inhibiting the expression of the target gene. This effect is called "RNA interference" or "RNAi." SIDR is complementary to a target sequence on a transcript and, after entering the cell, is loaded into the RNA-induced silencing complex (RISC). During this loading process, the sense strand is removed, leaving the antisense strand in the RISC, where it binds to its complementary site on the target RNA. The bound RNA is then cleaved by the nuclease activity of the RISC and can then be further degraded by cellular nucleases (see, for example, Non-Patent Document 1).

[0003] siRNAs composed of natural RNA nucleotides are rapidly degraded when administered in vivo and do not easily reach target organs. Therefore, chemically modified siRNAs must be used for therapeutic purposes. Typically, 2'-fluoro (2'-F) nucleotides and 2'-O-methyl (2'-O-Me) nucleotides are used as chemically modified nucleic acid units (modified nucleosides or modified nucleotides, which are phosphate adducts thereof) to enhance metabolic stability of siRNAs (see, for example, Non-Patent Documents 2 and 3).

[0004] Methylcarbamoylethylated nucleotides (2'-O-MCE nucleotides) have been reported as ribonucleotides with a modified oxygen atom at the 2'-position of the sugar moiety (see, for example, Patent Document 1 and Non-Patent Document 4). Furthermore, it has been reported that high nuclease resistance can be achieved by introducing an amino group or a heterocyclic group via an alkyl group into the nitrogen atom of the carbamoylethyl group (2'-O-R-ECE nucleotides) (see, for example, Patent Document 2 and Non-Patent Document 5). These nucleotides in which the oxygen atom at the 2'-position of the sugar moiety is modified via a carbamoylethyl group, such as 2'-O-MCE nucleotides and 2'-O-R-ECE nucleotides, are collectively referred to as 2'-O-XCE nucleotides.

[0005] International Publication No. WO 2007 / 102581 International Publication No. WO 2017 / 142054

[0006] Nature, 1998, 391, pp 806-811Nature Reviews Drug Discovery volume, 2019, 18, pp 421-446Molecular Therapy, 2018, 26, pp 708-717The Journal of Organic Chemistry, 2011, 76, pp 3042-3053Organic & Biomolecular Chemistry, 2019, 17, pp 4835-4842

[0007] There is a need for new technologies that further enhance metabolic stability while maintaining activity of chemically modified siRNA, or that improve activity and durability.

[0008] An object of the present invention is to provide chemically modified siRNAs that have enhanced metabolic stability while maintaining activity, or that have improved activity or durability.

[0009] The present inventors have discovered that partial substitution of 2'-O-XCE nucleotides in chemically modified siRNA can further enhance metabolic stability while maintaining activity, or improve activity and durability, and have completed the present invention. That is, the present invention encompasses the following aspects.

[0010] 1. A double-stranded RNA capable of inhibiting expression of a target RNA, the double-stranded RNA comprising a sense strand and an antisense strand, the antisense strand having 14-40 nucleotides and sufficient complementarity with the target RNA to mediate RNA interference, the sense strand having 14-40 nucleotides and complementarity with the antisense strand, and the double-stranded RNA comprising at least one 2'-O-XCE nucleotide.

[0011] 2. The double-stranded RNA according to 1., wherein the antisense strand comprises at least one 2'-O-XCE nucleotide, or the sense strand comprises at least one 2'-O-XCE nucleotide. 2-2. The double-stranded RNA according to 1., wherein the antisense strand comprises at least one 2'-O-XCE nucleotide. 2-3. The double-stranded RNA according to 1., wherein the sense strand comprises at least one 2'-O-XCE nucleotide. 2-4. The double-stranded RNA according to 1., wherein the antisense strand and the sense strand each independently comprise at least one 2'-O-XCE nucleotide.

[0012] 3. The double-stranded RNA according to any one of 1. to 2-4., wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-fluoro nucleotide. 4. The double-stranded RNA according to any one of 1. to 3., wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-O-Me nucleotide. 4-2. The double-stranded RNA according to any one of 1. to 4., wherein the nucleotides constituting the double-stranded RNA are selected from 2'-O-XCE nucleotides, 2'-O-Me nucleotides, and 2'-fluoro nucleotides. 5. The double-stranded RNA according to any one of 1. to 4-2., wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one deoxyribonucleotide. 5-2. 6. The double-stranded RNA according to any one of 1. to 5., wherein the nucleotides constituting the double-stranded RNA are selected from 2'-O-XCE nucleotides, 2'-O-Me nucleotides, 2'-fluoro nucleotides, and deoxyribonucleotides.

[0013] 6. The double-stranded RNA according to any one of 1. to 5-2., wherein the antisense strand contains a 2'-O-XCE nucleotide at at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end of the antisense strand hybridizing portion. 6-2. The double-stranded RNA according to any one of 1. to 6., wherein the antisense strand contains a 2'-fluoronucleotide or deoxyribonucleotide at position 2, counting from the 5' end of the antisense strand hybridizing portion. 6-3. The double-stranded RNA according to any one of 1. to 6-2., wherein the antisense strand contains 1 to 10 (preferably 1 to 9, more preferably 1, 2, 3, or 4) 2'-O-XCE nucleotides. 6-4. The double-stranded RNA according to any one of 1. to 6-3., wherein the antisense strand comprises a 2'-O-XCE nucleotide at at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridizing portion. 6-5. The double-stranded RNA according to any one of 1. to 6-4., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 1, counting from the 5' end of the antisense strand hybridizing portion. 6-6. The double-stranded RNA according to any one of 1. to 6-5., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 3, counting from the 5' end of the antisense strand hybridizing portion. 6-7. The double-stranded RNA according to any one of 1. to 6-6., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 4, counting from the 5'-end of the antisense strand hybridizing portion. 6-8. The double-stranded RNA according to any one of 1. to 6-7., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 5, counting from the 5'-end of the antisense strand hybridizing portion. 6-9. The double-stranded RNA according to any one of 1. to 6-8., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 6, counting from the 5'-end of the antisense strand hybridizing portion.6-10. The double-stranded RNA according to any one of 1. to 6-9., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 7, counting from the 5'-end of the antisense strand hybridizing portion. 6-11. The double-stranded RNA according to any one of 1. to 6-10., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 8, counting from the 5'-end of the antisense strand hybridizing portion. 6-12. The double-stranded RNA according to any one of 1. to 6-11., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 9, counting from the 5'-end of the antisense strand hybridizing portion. 6-13. The double-stranded RNA according to any one of 1. to 6-12., wherein the antisense strand comprises a 2'-O-XCE nucleotide at position 10, counting from the 5'-end of the antisense strand hybridizing portion. The double-stranded RNA according to any one of the preceding claims.

[0014] 7. The double-stranded RNA according to any one of 1. to 6-13., wherein the sense strand contains a 2'-O-XCE nucleotide at at least one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 3' end of the sense strand hybridizing portion. 7-2. The double-stranded RNA according to any one of 1. to 7., wherein the sense strand contains, independently, a 2'-O-Me nucleotide, a 2'-fluoro nucleotide, or a deoxyribonucleotide, at positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion. 7-3. The double-stranded RNA according to any one of 1. to 7-2., wherein the sense strand contains 2 to 10 (preferably 2 to 4, more preferably 2 or 4) 2'-O-XCE nucleotides. 7-4. The double-stranded RNA according to any one of 1. to 7-3., wherein the sense strand comprises a 2'-O-XCE nucleotide at at least one of positions 1, 2, 20, and 21, counting from the 3' end of the sense strand hybridizing portion. 7-5. The double-stranded RNA according to any one of 1. to 7-4., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 1, counting from the 3' end of the sense strand hybridizing portion. 7-6. The double-stranded RNA according to any one of 1. to 7-5., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 21, counting from the 3' end of the sense strand hybridizing portion. 7-7. The double-stranded RNA according to any one of 1. to 7-6., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 2, counting from the 3'-end of the sense strand hybridizing portion. 7-8. The double-stranded RNA according to any one of 1. to 7-7., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 20, counting from the 3'-end of the sense strand hybridizing portion. 7-9. The double-stranded RNA according to any one of 1. to 7-8., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the sense strand hybridizing portion. 7-10. The double-stranded RNA according to any one of 1. to 7-9., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 2, counting from the 5'-end of the sense strand hybridizing portion.7-11. The double-stranded RNA according to any one of 1. to 7-10., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the sense strand. 7-12. The double-stranded RNA according to any one of 1. to 7-11., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 2, counting from the 5'-end of the sense strand. 7-13. The double-stranded RNA according to any one of 1. to 7-12., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 1, counting from the 3'-end of the sense strand. 7-14. The double-stranded RNA according to any one of 1. to 7-13., wherein the sense strand comprises a 2'-O-XCE nucleotide at position 2, counting from the 3'-end of the sense strand.

[0015] 8. The 2'-O-XCE nucleotide is represented by the following formula (I): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group), X is a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR３ R ４ (The R ３ and R ４ each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ and n is a nucleotide having a partial structure represented by the following formula (I):

[0016] 9. The double-stranded RNA according to 8., wherein X is a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group). 10. The double-stranded RNA according to 8. or 9., wherein X is a methyl group. 11. X is a group represented by the following formula (Ia): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different. １ and R ２ 13. The double-stranded RNA according to 11., wherein Y is a hydrogen atom. ３ R ４ and the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 4-8 membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, or Y is a C2-9 aromatic heterocyclic group. 14. The double-stranded RNA according to 11 or 12., wherein Y is NR ３ R ４ and the R ３ and R ４ and form morpholine together with the nitrogen atom to which they are bonded. 15. The double-stranded RNA according to any one of 11 to 13, wherein Y is a pyridyl group, an imidazolyl group, or a benzimidazolyl group. 16. The double-stranded RNA according to any one of 11 to 15, wherein n is 2.

[0017] 17. The double-stranded RNA according to any one of 1. to 16., wherein the antisense strand contains 1, 2, 3, 4, 5, or 6 2'-fluoro nucleotides. 17-2. The double-stranded RNA according to any one of 1. to 17., wherein the antisense strand contains a 2'-fluoro nucleotide at at least one position selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. 17-3. The double-stranded RNA according to any one of 1. to 17-2., wherein the antisense strand contains a 2'-fluoro nucleotide at position 2, counting from the 5'-end of the antisense strand hybridizing portion. 17-4. The double-stranded RNA according to any one of 1. to 17-3., wherein the antisense strand contains a 2'-fluoro nucleotide at position 14, counting from the 5'-end of the antisense strand hybridizing portion. 17-5. The double-stranded RNA according to any one of 1. to 17-4., wherein the antisense strand contains a 2'-fluoro nucleotide at position 16, counting from the 5' end of the antisense strand hybridizing portion. 17-6. The double-stranded RNA according to any one of 1. to 17-5., wherein the antisense strand contains a 2'-fluoro nucleotide at position 6, counting from the 5' end of the antisense strand hybridizing portion.

[0018] 18. The double-stranded RNA according to any one of 1. to 17-6., wherein the sense strand contains 1, 2, 3, 4, or 5 2'-fluoro nucleotides. 18-2. The double-stranded RNA according to any one of 1. to 18., wherein the sense strand contains a 2'-fluoro nucleotide at at least one position selected from positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion. 18-3. The double-stranded RNA according to any one of 1. to 18-2., wherein the sense strand contains a 2'-fluoro nucleotide at position 15, counting from the 3' end of the sense strand hybridizing portion. 18-4. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a 2'-fluoro nucleotide at positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion. 18-5. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a deoxyribonucleotide at position 11, counting from the 3'-end of the sense strand hybridizing portion, and 2'-fluoronucleotides at positions 12 and 13. 18-6. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a deoxyribonucleotide at position 12, counting from the 3'-end of the sense strand hybridizing portion, and 2'-fluoronucleotides at positions 11 and 13. 18-7. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a deoxyribonucleotide at position 13, counting from the 3'-end of the sense strand hybridizing portion, and 2'-fluoronucleotides at positions 11 and 12. 18-8. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a 2'-O-Me nucleotide at position 11, counting from the 3'-end of the sense strand hybridizing portion, and 2'-fluoro nucleotides at positions 12 and 13. 18-9. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a 2'-O-Me nucleotide at position 12, counting from the 3'-end of the sense strand hybridizing portion, and 2'-fluoro nucleotides at positions 11 and 13.18-10. The double-stranded RNA according to any one of 1. to 18-3., wherein the sense strand contains a 2'-O-Me nucleotide at position 13 and 2'-fluoro nucleotides at positions 11 and 12, counting from the 3' end of the sense strand hybridizing portion.

[0019] 19. The double-stranded RNA according to any one of 1. to 18-10., wherein the sense strand comprises 7 to 18 2'-O-Me nucleotides. 19-2. The double-stranded RNA according to any one of 1. to 19., wherein the sense strand comprises 13, 14, 15, 16, 17, or 18 2'-O-Me nucleotides. 20. The double-stranded RNA according to any one of 1. to 19-2., wherein the antisense strand comprises 13 to 20 2'-O-Me nucleotides. 20-2. The double-stranded RNA according to any one of 1. to 20., wherein the antisense strand comprises 13 to 18 2'-O-Me nucleotides. 21. The double-stranded RNA according to any one of 1. to 20-2., wherein the sense strand comprises 0 to 6 deoxyribonucleotides. 21-2. The double-stranded RNA according to any one of 1. to 21., wherein the sense strand contains 0 or 1 deoxyribonucleotide. 22. The double-stranded RNA according to any one of 1. to 21-2., wherein the antisense strand contains 0 to 6 deoxyribonucleotides. 22-2. The double-stranded RNA according to any one of 1. to 22., wherein the antisense strand contains deoxyribonucleotides at 1 to 6 (preferably 2 to 6, 3 to 5) positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. 22-3. The double-stranded RNA according to any one of 1. to 22-2., wherein the antisense strand contains deoxyribonucleotides at 1 to 4 (preferably 2, 3, or 4) positions selected from positions 2, 5, 7, and 12, counting from the 5'-end of the antisense strand hybridizing portion. 22-4. The double-stranded RNA according to any one of 1. to 22-3., wherein the antisense strand contains deoxyribonucleotides at one or two positions selected from positions 5 and 7 counting from the 5' end of the antisense strand hybridizing portion. 22-5. The double-stranded RNA according to any one of 1. to 22., wherein the antisense strand does not contain deoxyribonucleotides.

[0020] 23. The double-stranded RNA according to any one of 1. to 22-5., wherein the double-stranded RNA contains at least one phosphorothioate bond. 23-2. The double-stranded RNA according to any one of 1. to 23., wherein the bonds connecting the nucleotides constituting the double-stranded RNA are each independently a phosphodiester bond or a modified phosphodiester bond (preferably a phosphorothioate bond). 24. The double-stranded RNA according to any one of 1. to 23-2., wherein the sense strand contains 1, 2, 3, or 4 phosphorothioate bonds. 25. The double-stranded RNA according to 1. to 24., wherein the sense strand contains a phosphorothioate bond at at least one position selected from the group consisting of between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, counting from the 5' end of the sense strand, and between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3' end of the sense strand. 25-2. The double-stranded RNA according to any one of 1. to 25., wherein the sense strand comprises phosphorothioate bonds between nucleotide positions 1 and 2, 2 and 3, counting from the 5' end of the sense strand, and between nucleotide positions 1 and 2, and 2 and 3, counting from the 3' end of the sense strand. 26. The double-stranded RNA according to any one of 1. to 25-2., wherein the antisense strand comprises 1, 2, 3, or 4 phosphorothioate bonds. 27. The double-stranded RNA according to any one of 1. to 26., wherein the antisense strand comprises phosphorothioate bonds between nucleotide positions 1 and 2, 2 and 3, counting from the 5' end of the antisense strand, and between nucleotide positions 1 and 2, and 2 and 3, counting from the 3' end of the antisense strand.

[0021] 28. The double-stranded RNA according to any one of 1. to 27., wherein the antisense strand comprises a 5'-phosphonate group at the 5'-end of the antisense strand. 29. The double-stranded RNA according to 28., wherein the 5'-phosphonate group is a 5'-vinylphosphonate (VP) group. 30. The double-stranded RNA according to 28. or 29., wherein the antisense strand comprises a 5'-vinylphosphonate (VP)-modified 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand. 31. The double-stranded RNA according to 28. or 29., wherein the antisense strand comprises a 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the antisense strand. 32. The double-stranded RNA according to 28., wherein the 5'-phosphonate group is a 5'-cyclopropanephosphonate (CPP) group. 33. The double-stranded RNA according to 28. or 32., wherein the antisense strand comprises a 5'-cyclopropanephosphonate (CPP)-modified 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand. 34. The double-stranded RNA according to 28. or 32., wherein the antisense strand comprises a 5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the antisense strand. 35. The double-stranded RNA according to 28., wherein the 5'-phosphonate group is a 5'-ethylphosphonate (EP) group. 36. The double-stranded RNA according to 28. or 35., wherein the antisense strand comprises a 5'-ethylphosphonate (EP)-modified 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand. 37. 28. The double-stranded RNA according to 28. or 35., wherein the antisense strand comprises a 5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide at position 1, counting from the 5' end of the antisense strand.

[0022] 38. The double-stranded RNA according to any one of 1. to 37., wherein the sense strand and the antisense strand are independently 19 to 25 (preferably 19 to 23) nucleotides in length. 38-2. The double-stranded RNA according to any one of 1. to 38., wherein the sense strand is 19 to 21 nucleotides in length and the antisense strand is 21 to 23 nucleotides in length. 39. The double-stranded RNA according to any one of 1. to 38-2., wherein the sense strand is 21 nucleotides in length. 40. The double-stranded RNA according to any one of 1. to 39., wherein the antisense strand is 23 nucleotides in length. 41. The double-stranded RNA according to any one of 1. to 40., wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. The double-stranded RNA according to any one of the preceding claims.

[0023] 42. The double-stranded RNA according to any one of 1. to 41., which comprises an overhang at the 3' end of the antisense strand. 42-2. The double-stranded RNA according to 42., wherein the overhang at the 3' end of the antisense strand is 2 nucleotides in length. 43. The double-stranded RNA according to any one of 1. to 42-2., which comprises a blunt end at the 5' end of the antisense strand.

[0024] 44. The double-stranded RNA according to any one of 1. to 43., further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site. 45. The double-stranded RNA according to 44., wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof. 46. The double-stranded RNA according to 44. or 45., wherein the functional molecule is selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, C5-30 unsaturated fatty acids, C5-30 alkyl groups, and C5-30 alkenyl groups. 46-2. The double-stranded RNA according to 46., wherein the functional molecule is a lipid selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, and C5-30 unsaturated fatty acids. 46-3. The double-stranded RNA according to 46., wherein the group derived from the functional molecule is a C5-30 alkyl group (preferably a C14-24 alkyl group, a C16-22 alkyl group, or a hexadecyl group). 47. The double-stranded RNA according to 44. or 45., wherein the functional molecule is a peptide or protein selected from the group consisting of receptor ligands and antibodies. 48. The double-stranded RNA according to 44. or 45., wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor. 48-2. The double-stranded RNA according to 44. or 45., wherein the group derived from the functional molecule is a group represented by the following formula: (wherein Rd is a hydroxy group or a thiol group (preferably a thiol group)). 48-3. The double-stranded RNA according to 48., wherein the group derived from the functional molecule is a ligand group represented by the following formula: (wherein Re each independently represents a hydroxy group or a thiol group (preferably a thiol group)). 48-4. The double-stranded RNA according to any one of 44. to 48-3., wherein the functional molecule is bound to the 5'-end or 3'-end of the sense strand or the antisense strand. 48-5. The double-stranded RNA according to any one of 44. to 48-3., wherein the functional molecule is bound to the 3'-end of the sense strand. 48-6. The double-stranded RNA according to any one of 44. to 48-3., wherein the functional molecule is bound to an internal nucleotide of the sense strand (preferably the oxygen atom at the 2'-position of the internal nucleotide). 48-7. The double-stranded RNA according to any one of 48. to 48-3., wherein the functional molecule is bound to an internal nucleotide of the antisense strand (preferably the oxygen atom at the 2'-position of the internal nucleotide).

[0025] 49. A pharmaceutical composition comprising the double-stranded RNA of any one of 1. to 48-7. and a pharmacologically acceptable carrier. 50. A method for regulating the function of a target RNA, comprising the step of contacting the double-stranded RNA of any one of 1. to 49. with a cell. 51. A method for regulating the function of a target RNA in a mammal, comprising the step of administering to the mammal the pharmaceutical composition of 49.. 52. A method for producing the double-stranded RNA of any one of 1. to 48-7. using a 2'-O-XCE nucleotide.

[0026] 53. A compound represented by the following formula (II): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IIa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], A １ is represented by the following formula (IIc): [Q １ and Q ２ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof. １ and Q ２ 53. The compound or salt thereof according to 53., wherein R is a hydrogen atom. １ and R ２ is a hydrogen atom, and the Y is NR ３ R ４ and the R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, and Q １ and Q ２ 56. The compound or salt thereof according to any one of 53. to 55., wherein the phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group.

[0027] 57. Formula (III): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３ represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IIIa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IIIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], B １ is represented by the following formula (IIIc): [Q ３ and Q ４ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof. 58. The compound represented by the formula (1) above, wherein X is a methyl group and Q ３ and Q ４ 57. The compound or salt thereof according to 57., wherein R is a hydrogen atom. １ and R ２ is a hydrogen atom, and the Y is NR ３ R ４ and the R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, and Q ３ and Q ４ 57. The compound according to 58., or a salt thereof, wherein is a hydrogen atom. 60. The compound according to any one of 57. to 59., or a salt thereof, wherein the phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group.

[0028] 61. Formula (IV): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３ represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IVa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IVb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], E １ is represented by the following formula (IVc): [Q ５ ~Q ８ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof. ５ ~Q ８ 61. The compound or salt thereof according to 61., wherein R is a hydrogen atom. １ and R ２ is a hydrogen atom, and the Y is NR ３ R ４ and the R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, and Q ５ ~Q ８ 64. The compound or salt thereof according to any one of 61. to 63., wherein the phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group.

[0029] The present invention provides chemically modified siRNAs that have enhanced metabolic stability while maintaining activity, or that have improved activity or durability.

[0030] The double-stranded RNA of the present invention can effectively regulate the expression of a target RNA and is useful as a nucleic acid pharmaceutical.

[0031]

[0033] Figure 1 shows a single crystal X-ray analysis ORTEP diagram of isomer A of compound 13.

[0034] Figure 2 shows the rat Sod1 gene expression level for each siRNA (si-0001 to si-0014) measured by quantitative real-time PCR in Evaluation Example 1.

[0035] Figure 3 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0015 to si-0027) measured by quantitative real-time PCR in Evaluation Example 1.

[0036] Figure 4 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0028 to si-0040) measured by quantitative real-time PCR in Evaluation Example 1.

[0037] Figure 5 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0041 to si-0053) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0054 to si-0066) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression level for each siRNA (si-0067 to si-0080) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression level for each siRNA (si-0067, si-0081 to si-0093) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression level for each siRNA (si-0067, si-0094 to si-0106) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression level for each siRNA (si-0067, si-0107 to si-0119) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression levels for each siRNA (si-0067, si-0120 to si-0132) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression levels for each siRNA (si-0001, i-0133 to si-0138) measured by quantitative real-time PCR in Evaluation Example 1. 1 shows the rat Sod1 gene expression levels for each siRNA (si-0001, si-0139 to si-0145) measured by quantitative real-time PCR in Evaluation Example 1.1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0146 to si-0157) measured by quantitative real-time PCR in Evaluation Example 2. 2 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0158 to si-0161) measured by quantitative real-time PCR in Evaluation Example 2. 3 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0162 to si-0169) measured by quantitative real-time PCR in Evaluation Example 2. 4 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0170 to si-0182) measured by quantitative real-time PCR in Evaluation Example 2. 5 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0183 to si-0190) measured by quantitative real-time PCR in Evaluation Example 2. 1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0191 to si-0197) measured by quantitative real-time PCR in Evaluation Example 2. 1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0198 to si-0208) measured by quantitative real-time PCR in Evaluation Example 2. 1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0209 to si-0211) measured by quantitative real-time PCR in Evaluation Example 3. 1 shows the rat Sod1 gene expression level for each siRNA (si-0001, si-0212 to si-0223) measured by quantitative real-time PCR in Evaluation Example 3. 1 shows the mouse Sod1 gene expression level in the lung for each siRNA (si-0224 to si-0227) measured by quantitative real-time PCR in Evaluation Example 6. 1 shows the expression level of mouse Sod1 gene in the lung for each siRNA (si-0224 to si-0225) by quantitative real-time PCR, as measured in Evaluation Example 7. FIG. 1 shows the expression level of mouse Sod1 gene in the cerebrum for each siRNA (si-0224 to si-0226 and si-0228) by quantitative real-time PCR, as measured in Evaluation Example 8.1 shows the mouse Sod1 gene expression level in the hippocampus for each siRNA (si-0224 to si-0226 and si-0228) measured by quantitative real-time PCR in Evaluation Example 8. 1 shows the mouse Sod1 gene expression level in the cerebellum for each siRNA (si-0224 to si-0226 and si-0228) measured by quantitative real-time PCR in Evaluation Example 8. 1 shows the mouse Sod1 gene expression level in the medulla oblongata for each siRNA (si-0224 to si-0226 and si-0228) measured by quantitative real-time PCR in Evaluation Example 8. 1 shows the mouse Ttr gene expression level for each siRNA (si-0229 to si-0241) measured by quantitative real-time PCR in Evaluation Example 9. 1 shows the mouse Ttr gene expression level for each siRNA (si-0229, si-0242 to si-0250) measured by quantitative real-time PCR in Evaluation Example 9. 1 shows the mouse Ttr gene expression level for each siRNA (si-0229, si-0251 to si-0259) measured by quantitative real-time PCR in Evaluation Example 9. 1 shows the mouse FXII gene expression level for each siRNA (si-0260 to si-0270) measured by quantitative real-time PCR in Evaluation Example 10. 1 shows the mouse FXII gene expression level for each siRNA (si-0260, si-0271 to si-0281) measured by quantitative real-time PCR in Evaluation Example 10. 1 shows the mouse FXII gene expression level for each siRNA (si-0260, si-0282 to si-0289) measured by quantitative real-time PCR in Evaluation Example 10. 1 shows the mouse serum TTR expression level for each siRNA (si-0290 to si-0297) measured by ELISA in Evaluation Example 11. 1 shows the mouse Ttr gene expression level for each siRNA (si-0298 to si-0305) measured by quantitative real-time PCR in Evaluation Example 12. 1 shows the mouse Ttr gene expression level for each siRNA (si-0306 to si-0312) measured by quantitative real-time PCR in Evaluation Example 12. 1 shows the mouse Ttr gene expression level for each siRNA (si-0313 to si-0321) measured by quantitative real-time PCR in Evaluation Example 13.1 shows the mouse Ttr gene expression level for each siRNA (si-0322 to si-0329) measured by quantitative real-time PCR in Evaluation Example 13. 1 shows the mouse Ttr gene expression level for each siRNA (si-0330 to si-0343) measured by quantitative real-time PCR in Evaluation Example 14. 1 shows the mouse Ttr gene expression level for each siRNA (si-0298, si-0344 to si-0349) measured by quantitative real-time PCR in Evaluation Example 15. 1 shows the mouse serum TTR expression level for each siRNA (si-0350 to si-0351) measured by ELISA in Evaluation Example 16. 1 shows the rat Sod1 gene expression level for each siRNA (si-0209, si-0216, si-0352 to si-0362) measured by quantitative real-time PCR in Evaluation Example 17. 1 shows the rat Sod1 gene expression level for each siRNA (si-0209, si-0216, si-0363 to si-0373) measured by quantitative real-time PCR in Evaluation Example 17. 1 shows the rat Sod1 gene expression level for each siRNA (si-0209, si-0216, si-0374 to si-0383) measured by quantitative real-time PCR in Evaluation Example 18. 1 shows the rat Sod1 gene expression level for each siRNA (si-0209, si-0216, si-0384 to si-0394) measured by quantitative real-time PCR in Evaluation Example 18. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0253, si-0395 to si-0403) measured by quantitative real-time PCR in Evaluation Example 19. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0253, si-0404 to si-0416) measured by quantitative real-time PCR in Evaluation Example 19. 2 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0411 to si-0412) measured by quantitative real-time PCR in Evaluation Example 19. 3 shows the mouse FXII gene expression level for each siRNA (si-0282, si-0417 to si-0419) measured by quantitative real-time PCR in Evaluation Example 20.1 shows the mouse FXII gene expression level for each siRNA (si-0282, si-0284, si-0420 to si-0425) measured by quantitative real-time PCR in Evaluation Example 20. 1 shows the mouse Ttr gene expression level for each siRNA (si-0298, si-0347, si-0426 to si-0429) measured by quantitative real-time PCR in Evaluation Example 21. 1 shows the mouse Ttr gene expression level for each siRNA (si-0298, si-0430 to si-0436) measured by quantitative real-time PCR in Evaluation Example 21. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0437 to si-0448) measured by quantitative real-time PCR in Evaluation Example 22. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0449 to si-0453) measured by quantitative real-time PCR in Evaluation Example 23. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0454 to si-0460) measured by quantitative real-time PCR in Evaluation Example 24. 1 shows the mouse Ttr gene expression level for each siRNA (si-0251, si-0461 to si-0467) measured by quantitative real-time PCR in Evaluation Example 25. 1 shows the mouse Ttr gene expression level for each siRNA (si-0298, si-0468 to si-0470) measured by quantitative real-time PCR in Evaluation Example 26. 1 shows the rat Sod1 gene expression levels for each siRNA (si-0209, si-0216, si-0220, si-0471 to si-0480) measured by quantitative real-time PCR in Evaluation Example 27. 1 shows the rat Sod1 gene expression levels for each siRNA (si-0209, si-0481 to si-0486) measured by quantitative real-time PCR in Evaluation Example 29. 1 shows the mouse serum TTR expression levels for each siRNA (si-0488, si-00489, si-0497 to si-0499) measured by ELISA in Evaluation Example 30. 1 shows the mouse serum TTR expression levels for each siRNA (si-0488, si-00489, si-0493 to si-0495, si-0500) measured by ELISA in Evaluation Example 30.1 shows the TTR expression levels in mouse serum for each siRNA (si-0488, si-00489, si-0490 to si-0492, si-0496) by ELISA, measured in Evaluation Example 30. 1 shows the nuclease resistance of oligonucleotides, measured in Evaluation Example 31. 1 shows the nuclease resistance of oligonucleotides, measured in Evaluation Example 32.

[0032] Terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Each term used in this specification is explained below. In this specification, each term has the same meaning whether used alone or in combination with other terms, unless otherwise specified. It is understood that both the summary above and the detailed description below are merely exemplary and explanatory and do not limit the claimed invention. In this specification, the use of the singular form includes the plural form unless specifically stated otherwise.

[0033] "n-" means normal, "i-" means iso, "s-" means secondary, "t-" means tertiary, "o-" means ortho, "m-" means meta, and "p-" means para. "Ph" means phenyl, "Me" means methyl, "Bu" means butyl, "Ac" means acetyl, and "DMTr" means dimethoxytrityl.

[0034] "Halogen atom" means a fluorine atom, chlorine atom, bromine atom or iodine atom.

[0035] The term "C1-6 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 6 carbon atoms. Examples of C1-6 alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, and isohexyl. Similarly, the term "C1-4 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 4 carbon atoms. Similarly, the term "C1-3 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 1 to 3 carbon atoms. Similarly, the term "C5-30 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 5 to 30 carbon atoms. Examples of C5-30 alkyl groups include n-pentyl, n-hexyl, isohexyl, n-heptyl, decyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, and triacontyl. Similarly, a "C14-24 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 14 to 24 carbon atoms. Similarly, a "C16-22 alkyl group" refers to a monovalent linear or branched saturated aliphatic hydrocarbon group having 16 to 22 carbon atoms.

[0036] The term "halo C1-6 alkyl group" refers to the above "C1-6 alkyl group" in which at least one hydrogen atom at any position is substituted with the above "halogen atom".

[0037] The term "C2-6 alkenyl group" refers to a monovalent linear or branched, unsaturated aliphatic hydrocarbon group having 2 to 6 carbon atoms and containing at least one carbon-carbon double bond. Examples of C2-6 alkenyl groups include vinyl, allyl, propenyl, isopropenyl, butenyl, isobutenyl, butadienyl, 3-methyl-2-butenyl, pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, and hexadienyl. Similarly, the term "C5-30 alkenyl group" refers to a monovalent linear or branched, unsaturated aliphatic hydrocarbon group having 5 to 30 carbon atoms and containing at least one carbon-carbon double bond. Examples of C5-30 alkenyl groups include pentenyl, isopentenyl, pentadienyl, hexenyl, isohexenyl, hexadienyl, decenyl, tetradecenyl, hexadecenyl, octadecenyl, icosenyl, docosenyl, tetracosenyl, and triacontenyl groups.

[0038] The term "C1-6 alkoxy group" refers to a group in which the above-mentioned "C1-6 alkyl group" is bonded to an oxy (-O-) group. Examples of C1-6 alkoxy groups include methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy, isobutoxy, s-butoxy, n-pentyloxy, isopentyloxy, and n-hexyloxy.

[0039] The term "C2-6 alkenyloxy group" refers to a group in which the above "C2-6 alkenyl group" is bonded to an oxy (-O-) group. Examples of the C2-6 alkenyloxy group include a vinyloxy group, an allyloxy group, a propenyloxy group, an isopropenyloxy group, a butenyloxy group, an isobutenyloxy group, a butadienyloxy group, a 3-methyl-2-butenyloxy group, a pentenyloxy group, an isopentenyloxy group, a pentadienyloxy group, a hexenyloxy group, an isohexenyloxy group, and a hexadienyloxy group.

[0040] The term "C1-6 alkylcarbonyl group" refers to a group in which the above-mentioned "C1-6 alkyl group" is bonded to a carbonyl (-C(=O)-) group.

[0041] The term "C1-6 alkoxycarbonyl group" refers to a group in which the "C1-6 alkoxy group" is bonded to a carbonyl (-C(=O)-) group, and the term "C2-6 alkenyloxycarbonyl group" refers to a group in which the "C2-6 alkenyloxy group" is bonded to a carbonyl (-C(=O)-) group.

[0042] The term "C2-20 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 2 to 20 carbon atoms. The term "C8-12 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 8 to 12 carbon atoms, among the aforementioned "C2-20 alkylene groups". The term "C2-6 alkylene group" refers to a divalent group of a linear or branched saturated aliphatic hydrocarbon having from 2 to 6 carbon atoms, among the aforementioned "C2-20 alkylene groups", and examples thereof include an ethylene (ethanediyl) group, a propylene group, a propane-1,3-diyl (trimethylene) group, a propane-2,2-diyl (isopropylidene) group, a 2,2-dimethyl-propane-1,3-diyl group, a hexane-1,6-diyl (hexamethylene) group, and a 3-methylbutane-1,2-diyl group.

[0043] The term "C2-20 alkenylene group" refers to a divalent linear or branched unsaturated aliphatic hydrocarbon group containing 2 to 20 carbon atoms and at least one carbon-carbon double bond.

[0044] The term "C1-6 alkylamino group" includes mono-C1-6 alkylamino groups and di-C1-6 alkylamino groups. The term "mono-C1-6 alkylamino group" refers to a group in which one of the above-mentioned "C1-6 alkyl groups" is bonded to an amino group. Examples of mono-C1-6 alkylamino groups include methylamino group, ethylamino group, n-propylamino group, isopropylamino group, n-butylamino group, n-pentylamino group, and n-hexylamino group. The term "di-C1-6 alkylamino group" refers to a group in which two of the above-mentioned "C1-6 alkyl groups" are bonded to an amino group. The two alkyl groups may be the same or different. Examples of the di-C alkylamino group include a dimethylamino group, a diethylamino group, an N,N-diisopropylamino group, an N-methyl-N-ethylamino group, an N-isopropyl-N-methylamino group, an N-n-butyl-N-methylamino group, an N-tert-butyl-N-methylamino group, an N-methyl-N-pentylamino group, an N-n-hexyl-N-methylamino group, and an N-isopropyl-N-ethylamino group.

[0045] The term "C1-6 alkylaminocarbonyl group" means a group in which the above "C1-6 alkylamino group" is bonded to a carbonyl group.

[0046] The term "C1-6 alkylcarbonyloxy group" means a group in which the above "C1-6 alkylcarbonyl group" is bonded to an oxy group.

[0047] The term "C1-6 alkylcarbonylamino group" means a group in which one "C1-6 alkylcarbonyl group" as defined above is bound to an amino group.

[0048] The term "C1-6 alkoxycarbonylamino group" means a group in which one "C1-6 alkoxycarbonyl group" as defined above is bonded to an amino group.

[0049] The term "aryl group" refers to a monovalent group formed by removing one hydrogen atom at any position from a monocyclic or bicyclic aromatic hydrocarbon ring in which all atoms constituting the ring are carbon atoms, and specific examples include "C aryl groups" such as a phenyl group and a naphthyl group.

[0050] The term "aralkyl group" refers to a monovalent group in which a hydrogen atom at any position of the aforementioned "C1-6 alkyl group" is replaced by the aforementioned "C6-10 aryl group". The term "C7-10 aralkyl group" refers to a monovalent group in which a hydrogen atom at any position of the aforementioned "C1-4 alkyl group" is replaced by a phenyl group.

[0051] The term "3- to 11-membered nitrogen-containing non-aromatic heterocycle" refers to a monocyclic, fused polycyclic (in such a fused polycyclic, a non-aromatic ring may be fused to a non-aromatic ring or an aromatic ring), bridged ring, or spirocyclic non-aromatic heterocyclic compound containing at least one nitrogen atom and having 3 to 11 atoms constituting the ring, and examples thereof include azetidine, pyrrolidine, 2-oxopyrrolidine, piperidine, 3-oxopiperidine, piperazine, morpholine, thiomorpholine, homomorpholine, and homopiperazine.

[0052] The term "C2-9 aromatic heterocyclic group" refers to a monovalent group formed by removing one hydrogen atom at any position from an aromatic monocyclic or fused ring compound having 2 to 9 carbon atoms in the ring and one or more identical or different heteroatoms selected from oxygen atoms, sulfur atoms, and nitrogen atoms in the ring. Examples of C2-9 aromatic heterocycles include purinyl, pyrimidinyl, thienyl, furyl, isobenzofuranyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, isoxazolyl, oxadiazolyl, pyridyl, pyridazinyl, pyrazinyl, indolizinyl, indolyl, isoindolyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl, quinazolinyl, pteridinyl, benzofuranyl, benzothienyl, and benzimidazolyl groups.

[0053] The term "5- to 10-membered heterocyclic group" refers to a monovalent group obtained by removing one hydrogen atom at any position from a monocyclic or fused polycyclic aromatic heterocyclic compound having 5 to 10 atoms constituting the ring and containing 1 to 5 heteroatoms (the heteroatoms refer to nitrogen atoms, oxygen atoms, or sulfur atoms, and when there are two or more heteroatoms, they may be the same or different) among the atoms constituting the ring.

[0054] Examples of the monocyclic "5- to 10-membered heterocyclic group" include a 2-thienyl group, a 3-thienyl group, a 2-furyl group, a 3-furyl group, a 2-pyranyl group, a 3-pyranyl group, a 4-pyranyl group, a 1-pyrrolyl group, a 2-pyrrolyl group, a 3-pyrrolyl group, a 1-imidazolyl group, a 2-imidazolyl group, a 4-imidazolyl group, a 1-pyrazolyl group, a 3-pyrazolyl group, a 4-pyrazolyl group, a 5-pyrazolyl group, 2-thiazolyl group, 4-thiazolyl group, 5-thiazolyl group, 3-isothiazolyl group, 4-isothiazolyl group, 5-isothiazolyl group, 1,2,4-triazol-1-yl group, 1,2,4-triazol-3-yl group, 1,2,4-triazol-5-yl group, 1,2,3-triazol-1-yl group, 1,2,3-triazol-4-yl group, 1,2,3-triazolyl group Examples thereof include an oxazol-5-yl group, a 2-oxazolyl group, a 4-oxazolyl group, a 5-oxazolyl group, a 3-isoxazolyl group, a 4-isoxazolyl group, a 5-isoxazolyl group, a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-pyrazinyl group, a 2-pyrimidinyl group, a 4-pyrimidinyl group, a 5-pyrimidinyl group, a 3-pyridazinyl group, a 4-pyridazinyl group, a 1,3,4-oxadiazol-2-yl group, a 1,3,4-thiadiazol-2-yl group, a 1,2,4-oxadiazol-3-yl group, a 1,2,4-oxadiazol-5-yl group, a 1,2,4-thiadiazol-3-yl group, a 1,2,4-thiadiazol-5-yl group, a 1,2,5-oxadiazol-3-yl group, and a 1,2,5-thiadiazol-3-yl group.

[0055] Examples of the fused polycyclic "5- to 10-membered heterocyclic group" include a 2-benzofuranyl group, a 3-benzofuranyl group, a 4-benzofuranyl group, a 5-benzofuranyl group, a 6-benzofuranyl group, a 7-benzofuranyl group, a 1-isobenzofuranyl group, a 4-isobenzofuranyl group, a 5-isobenzofuranyl group, a 2-benzothienyl group, a 3-benzothienyl group, a 4-benzothienyl group, a 5-benzothienyl group, a 6-benzothienyl group, a 7-benzothienyl group, a 1-isobenzothienyl group, a 4-isobenzothienyl group, a 5-isobenzothienyl group, a 2-benzothiazolyl group, a 4- ... azothiazolyl group, a 5-benzothiazolyl group, a 6-benzothiazolyl group, a 7-benzothiazolyl group, a 2-chromenyl group, a 3-chromenyl group, a 4-chromenyl group, a 5-chromenyl group, a 6-chromenyl group, a 7-chromenyl group, an 8-chromenyl group, a 1-indolizinyl group, a 2-indolizinyl group, a 3-indolizinyl group, a 5-indolizinyl group, a 6-indolizinyl group, a 7-indolizinyl group, an 8-indolizinyl group, a 1-isoindolyl group, a 2-isoindolyl group, a 4-isoindolyl group, a 5-isoindolyl group, a 1-indolyl group, a 2-indolyl group, a 3- Indolyl group, 4-indolyl group, 5-indolyl group, 6-indolyl group, 7-indolyl group, 1-indazolyl group, 2-indazolyl group, 4-indazolyl group, 5-indazolyl group, 6-indazolyl group, 7-indazolyl group, 2-purinyl group, 6-purinyl group, 7-purinyl group, 8-purinyl group, 2-quinolyl group, 3-quinolyl group, 4-quinolyl group, 5-quinolyl group, 6-quinolyl group, 7-quinolyl group, 8-quinolyl group, 1-isoquinolyl group, 3-isoquinolyl group, 4-isoquinolyl group, 5-isoquinolyl group, 6-isoquinolyl group, 7-isoquinolyl group a quinolyl group, an 8-isoquinolyl group, a 1-phthalazinyl group, a 5-phthalazinyl group, a 6-phthalazinyl group, a 2,7-naphthyridin-1-yl group, a 2,7-naphthyridin-3-yl group, a 2,7-naphthyridin-4-yl group, a 2,6-naphthyridin-1-yl group, a 2,6-naphthyridin-3-yl group, a 2,6-naphthyridin-4-yl group, a 1,8-naphthyridin-2-yl group, a 1,8-naphthyridin-3-yl group, a 1,8-naphthyridin-4-yl group, a 1,7-naphthyridin-2-yl group, a 1,7-naphthyridin-3-yl group, a 1,7-naphthyridin-4-yl group,7-naphthyridin-5-yl group, 1,7-naphthyridin-6-yl group, 1,7-naphthyridin-8-yl group, 1,6-naphthyridin-2-yl group, 1,6-naphthyridin-3-yl group, 1,6-naphthyridin-4-yl group, 1,6-naphthyridin-5-yl group, 1,6-naphthyridin-7-ynyl group, 1,6-naphthyridin-8-yl group, 1,5-naphthyridin-2-yl group, 1,5-naphthyridin-3-yl group, 1,5-naphthyridin-4-yl group , 2-quinoxalinyl group, 5-quinoxalinyl group, 6-quinoxalinyl group, 2-quinazolinyl group, 4-quinazolinyl group, 5-quinazolinyl group, 6-quinazolinyl group, 7-quinazolinyl group, 8-quinazolinyl group, 3-cinnolinyl group, 4-cinnolinyl group, 5-cinnolinyl group, 6-cinnolinyl group, 7-cinnolinyl group, 8-cinnolinyl group, 2-pteridinyl group, 4-pteridinyl group, 6-pteridinyl group and 7-pteridinyl group.

[0056] "Oxo" refers to a group (=O) in which an oxygen atom is substituted via a double bond. When oxo is substituted on a carbon atom, it combines with said carbon atom to form a carbonyl. "Thioxo" refers to a group (=S) in which a sulfur atom is substituted via a double bond. When thioxo is substituted on a carbon atom, it combines with said carbon atom to form a thiocarbonyl.

[0057] The terms "protected hydroxy group," "protected amino group," and "protected sulfanyl group" mean a hydroxy group protected with a hydroxy-protecting group, an amino group protected with an amino-protecting group, and a sulfanyl group protected with a sulfanyl-protecting group, respectively. The hydroxy-protecting group, amino-protecting group, and sulfanyl-protecting group are not particularly limited as long as they are stable during oligonucleotide synthesis, and examples thereof include protecting groups described in Protective Groups in Organic Synthesis, 4th Edition, by T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006), which are well known to those skilled in the art, and the like.

[0058] For example, examples of the "hydroxy-protecting group" include ether-based protecting groups such as C1-6 alkyl (e.g., methyl, ethyl, t-butyl, etc.), triarylmethyl (e.g., triphenylmethyl (trityl), monomethoxytrityl, dimethoxytrityl (DMTr), trimethoxytrityl, etc.); acetal-based protecting groups such as methoxymethyl, methylthiomethyl, methoxyethyl, benzyloxymethyl, 2-tetrahydropyranyl, ethoxyethyl, etc.; acyl-based protecting groups such as acyl (e.g., formyl, acetyl, pivaloyl, benzoyl, etc.); tri(C1-6 alkyl)silyl (e.g., trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, dimethylisopropylsilyl, etc.); silyl, etc.), (C1-6 alkyl)diarylsilyl (for example, t-butyldiphenylsilyl, diphenylmethylsilyl, etc.), triarylsilyl (for example, triphenylsilyl, etc.), tribenzylsilyl, [(triisopropylsilyl)oxy]methyl (Tom group), and other silyl protecting groups; 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep group), 9-phenylxanthen-9-yl (Pixyl group), 9-(p-methoxyphenyl)xanthen-9-yl (MOX group), acyloxymethyl (for example, methyl substituted with a C1-6 alkylcarbonyloxy group, specifically, pivaloyloxymethyl, acetyloxymethyl, etc.), and the like.

[0059] For example, the "amino-protecting group" may be an amide-based protecting group such as acyl (for example, formyl, acetyl, propionyl, pivaloyl (Pv), tigloyl, etc.), haloacyl (for example, fluoroacetyl, difluoroacetyl, trifluoroacetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, etc.), arylcarbonyl (for example, benzoyl, p-bromobenzoyl, p-nitrobenzoyl, 2,4-dinitrobenzoyl, etc.); C alkoxycarbonyl (for example, methoxycarbonyl, ethoxycarbonyl, n-propoxycarbonyl, i-propoxycarbonyl, n-butoxycarbonyl, i-butoxycarbonyl, t-butoxycarbonyl (Boc), t- amyloxycarbonyl, and the like, preferably Boc, and the like), C2-6 alkenyloxycarbonyl (for example, vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc), and the like), tri(C1-3 alkyl)silylethoxycarbonyl (for example, 2-(trimethylsilyl)ethoxycarbonyl (Teoc), and the like), haloC1-6 alkoxycarbonyl (for example, 2,2,2-trichloroethoxycarbonyl (Troc), and the like), aryloxycarbonyl (for example, benzyloxycarbonyl (Z or Cbz), p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl (Moz), and the like); carbamate-based protecting groups such as C2-6 alkenyloxycarbonyl (for example, vinyloxycarbonyl (Voc), allyloxycarbonyl (Alloc), and the like), tri(C1-3 alkyl)silylethoxycarbonyl (for example, 2-(trimethylsilyl)ethoxycarbonyl (Teoc)), haloC1-6 alkoxycarbonyl (for example, 2,2,2-trichloroethoxycarbonyl (Troc)), and aryloxycarbonyl (for example, benzyloxycarbonyl (Z or Cbz), p-nitrobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl (Moz), and the like);and sulfonamide-based protecting groups such as alkylsulfonyl (for example, methanesulfonyl (Ms), ethanesulfonyl, etc.), arylsulfonyl (for example, benzenesulfonyl, p-toluenesulfonyl (Ts), p-chlorobenzenesulfonyl, p-methoxybenzenesulfonyl (MBS), m-nitrobenzenesulfonyl, o-nitrobenzenesulfonyl, p-nitrobenzenesulfonyl, 2,4-nitrobenzenesulfonyl, 2,6-dimethoxy-4-methylbenzenesulfonyl (iMds), 2,6-dimethyl-4-methoxybenzenesulfonyl (Mds), 2,4,6-trimethoxybenzenesulfonyl (Mtb), 2,3,5,6-tetramethyl-4-methoxybenzenesulfonyl (Mte), 2,3,6-trimethyl-4-methoxybenzenesulfonyl (Mtr), 2,4,6-trimethylbenzenesulfonyl (Mts), pentamethylbenzenesulfonyl (Pme), etc.);

[0060] For example, examples of the "sulfanyl protecting group" include ether-based protecting groups such as benzyl, p-methoxybenzyl, p-acetoxybenzyl, p-nitrobenzyl, 4-picolyl, 9-fluorenylmethyl, di- or triarylmethyl (for example, diphenylmethyl, triphenylmethyl (trityl), bis(4-methoxyphenyl)methyl, etc.); thioacetal-based protecting groups such as methoxymethyl, isobutoxymethyl, benzylthiomethyl, 2-tetrahydropyranyl, etc.; and thioester-based protecting groups such as acyl (for example, acetyl, benzoyl, etc.). In this specification, a sulfanyl group may be referred to as a thiol group.

[0061] For the protection and deprotection of the "hydroxy-protecting group," "amino-protecting group," and "sulfanyl-protecting group" in the present invention, reference can also be made to Protective Groups in Organic Synthesis, 4th Edition, T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006), etc.

[0062] The phosphorus-containing group refers to a group containing a phosphorus atom and useful for forming an internucleoside bond, including a phosphodiester structure or a phosphorothioate structure. Examples of the phosphorus-containing group include phosphorus-containing groups known in the art, such as groups derived from phosphoramidites, H-phosphonates, phosphate diesters, and phosphate triesters.

[0063] Specifically, the compound represented by the formula (Z ３ -1) to the formula (Z ３ -3) (wherein the phosphorus atom is bonded to the sugar moiety of the nucleotide via an oxygen atom). In the formula, R Ｘ１ and R Ｘ２ are each independently a C1-6 alkyl group, and the alkyl group is unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a cyano group, a C6-10 aryl group, and a C2-9 aromatic heterocyclic group.

[0064] In the formula, R Ｘ３ is an oxygen atom or a sulfur atom, and R Ｘ４ are each independently a hydrogen atom, a hydroxy protecting group, a C1-6 alkyl group or a C6-10 aryl group, the alkyl group being unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a cyano group, a C6-10 aryl group and a C2-9 aromatic heterocyclic group, and the aryl group being unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a C1-6 alkyl group and a cyano group.

[0065] In the formula, R Ｘ５ is an oxygen atom or a sulfur atom. Ｘ６ is a hydrogen atom, a hydroxy protecting group, or a C6-10 aryl group.

[0066] The phosphorus-containing group is preferably of the formula (Z ３−4) to the formula (Z ３ -6). In the formula, R Ｘ１ is a C1-6 alkyl group or a C1-6 alkyl group substituted with a cyano group, and R Ｘ２ is a C1-6 alkyl group. The phosphorus-containing group is more preferably a cyanoethoxy(diisopropylamino)phosphino group (formula: -P(OC ２ H ４ CN)(N(i-Pr) ２ ) or a hydroxyphosphinyl group (a group represented by the formula: —P(═O)H(OH)).

[0067] The term "phosphonate group" refers to a monovalent group containing one phosphorus-carbon single bond, one phosphorus-oxygen double bond, and two phosphorus-oxygen single bonds. The term "5'-phosphonate group" refers to the phosphonate group located at the 5'-position of an oligonucleotide. The 5'-phosphonate group is preferably located at the 5'-position of the 5'-end of the oligonucleotide. The bond between the nucleotide and the phosphonate group is preferably such that the oxygen atom at the 5'-position of the nucleotide is replaced with a carbon atom, and the carbon atom is bonded to the phosphorus atom. In this specification, the phosphonate group is encompassed by modified phosphate groups.

[0068] "RNA interference" means that the introduction of double-stranded RNA degrades a target RNA that has a sequence homologous to the introduced double-stranded RNA, thereby inhibiting the expression of the target RNA. "Mediating RNA interference" is understood to mean having the ability to degrade a target RNA in a sequence-specific manner and inhibit the expression of the target RNA by the introduction of double-stranded RNA.

[0069] "siRNA" refers to a double-stranded RNA that generates RNA interference by hybridizing an antisense strand and a sense strand that can hybridize with a target RNA. The antisense strand and the sense strand are, for example, 14 to 40 nucleotides long, 14 to 20 nucleotides long, and preferably 19 to 23 nucleotides long. The antisense strand is complementary to the target RNA and can be understood as a strand that is mainly composed of RNA, which may be modified. The sense strand is complementary to part or all of the antisense strand and can be understood as a strand that is mainly composed of RNA, which may be modified. The mechanism of action of RNA interference is not clear, but is thought to be as follows: After entering the cell, the antisense strand is loaded into the RNA-induced silencing complex (RISC). During this loading process, the sense strand is removed, and the antisense strand remains in the RISC, where it binds to its complementary site on the target RNA. The bound RNA is then cleaved by the nuclease activity of the RISC and can then be further degraded by cellular nucleases.

[0070] "Target RNA" refers to mRNA, mRNA precursor, or ncRNA, and includes mRNA transcribed from genomic DNA encoding a target gene, mRNA without base modifications, unspliced ​​mRNA precursor, and ncRNA. The "target RNA" whose expression is inhibited by RNA interference is not particularly limited, and examples include RNA associated with genes whose expression is increased in various diseases. "Target RNA" may be any RNA synthesized by DNA-dependent RNA polymerase, and is preferably mRNA or mRNA precursor. More preferably, it is mammalian mRNA, and even more preferably, it is human mRNA.

[0071] "Hybridize" refers to the act of forming a double strand between oligonucleotides or groups derived from oligonucleotides containing complementary sequences, and the phenomenon in which oligonucleotides or groups derived from oligonucleotides containing complementary sequences form a double strand. The antisense strand and the sense strand hybridize.

[0072] "Sequence portion" refers to a partial structure of an oligonucleotide chain. For example, a sequence portion containing a nucleotide is a partial structure of a region of an oligonucleotide chain that contains the nucleotide.

[0073] "Hybridizing portion" refers to the complementary sequence portion that forms the double strand. "Sense strand hybridizing portion" refers to a region of the sense strand that hybridizes with the antisense strand. "Antisense strand hybridizing portion" refers to a region of the antisense strand that hybridizes with the sense strand. The sense strand contained in the double-stranded RNA of the present invention contains a sense strand hybridizing portion, and the antisense strand contains an antisense strand hybridizing portion. The sense strand hybridizing portion and the antisense strand hybridizing portion are complementary to each other and form a double strand.

[0074] "Complementary" or "having complementarity" means that two nucleobases can form Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (such as Hoogsteen base pairs) through hydrogen bonds. Two oligonucleotides or oligonucleotide-derived groups can "hybridize" if their sequences are complementary. While two oligonucleotides or oligonucleotide-derived groups do not need to be completely complementary to hybridize, the degree of complementarity required for two oligonucleotides or oligonucleotide-derived groups to hybridize is preferably 70% or greater, more preferably 80% or greater, and even more preferably 90% or greater (e.g., 95% or greater, 96% or greater, 97% or greater, 98% or greater, 99% or greater, or 100%). Sequence complementarity can be determined using a computer program that automatically identifies subsequences of oligonucleotides. For example, OligoAnalyzer is one such software program provided by Integrated DNA Technologies. This program is also available on the website. Those skilled in the art can easily determine the conditions (temperature, salt concentration, etc.) under which two oligonucleotides or oligonucleotide-derived groups can hybridize. Furthermore, those skilled in the art can easily design an antisense strand complementary to a target RNA, for example, by using a BLAST program based on information about the nucleotide sequence of the target RNA. For details of the BLAST program, see Proceedings of the National Academy of Sciences of the United States of America, 1990, 87, pp. 2264-2268; ibid., 1993, 90, pp. 5873-5877; and Journal of Molecular Biology, 1990, 215, pp. 403-410.The "sufficient complementarity with the target RNA to mediate RNA interference" is not particularly limited as long as it is sufficient complementarity with the target RNA to mediate RNA interference, but is, for example, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%).

[0075] "Nucleotide" refers to a molecule that can be a building block of a nucleic acid (oligonucleotide), and typically has a base and / or a sugar as a component. A nucleotide is composed of, for example, a sugar, a base, and a phosphate. The sugar, base, and phosphate may be modified. Nucleotides include ribonucleotides, deoxyribonucleotides, and sugar-modified nucleotides, which will be described later. Nucleotides also include molecules that do not have a base and are composed of a sugar and a phosphate, as well as modified versions of these.

[0076] "Nucleoside" refers to a molecule that can be a structural unit of a nucleic acid (oligonucleotide), and typically has a base as a constituent element. A nucleoside is, for example, composed of a sugar and a base, and represents a partial structure of the aforementioned "nucleotide" that does not contain phosphate or modified phosphate. A "nucleoside structure" refers to a structural unit that links adjacent nucleosides in an oligonucleotide via a phosphodiester bond or a modified phosphodiester bond. In this specification, the term "nucleoside structure" is used to represent a partial structure that does not contain a phosphodiester bond or a modified phosphodiester bond.

[0077] An "oligonucleotide" refers to a molecule having a polymerized structure of one or more of the above nucleotides. When an "oligonucleotide" is composed of one nucleotide, the oligonucleotide can be referred to as a "nucleotide." The nucleotides contained in the double-stranded RNA of the present invention are each independently linked to each other by a phosphodiester bond or a modified phosphodiester bond described below. The 3'-terminal nucleotide of each strand of the oligonucleotide contained in the double-stranded RNA of the present invention preferably has a hydroxy group, a phosphate group, or a modified phosphate group, more preferably a hydroxy group, and usually a hydroxy group, at its 3'-position. The 5'-terminal nucleotide of each strand of the oligonucleotide contained in the double-stranded RNA preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 5'-position.

[0078] "Internucleotide linkage" means a group or bond that forms a covalent bond between adjacent nucleosides in an oligonucleotide. Internucleotide linkages include phosphodiester linkages or modified phosphodiester linkages (e.g., phosphorothioate linkages). Internucleotide linkages may also be referred to as internucleoside linkages.

[0079] The term "group derived from an oligonucleotide" refers to a group obtained by removing a hydrogen atom, a hydroxy group, or the like from a hydroxy group at at least one of the 3'- and 5'-ends of the oligonucleotide, and is indirectly covalently linked to another group (e.g., a group derived from another oligonucleotide) by forming a phosphodiester bond or a modified phosphodiester bond. The hydroxy group at the 3'- or 5'-end includes a hydroxy group contained in a phosphate group. For example, a group obtained by removing a hydrogen atom from the hydroxy group at the 3'-end of an oligonucleotide and a group obtained by removing a hydroxy group from the phosphate group at the 5'-end of another oligonucleotide form a phosphodiester bond or a modified phosphodiester bond.

[0080] Similarly, a "group derived from" a molecule means a group obtained by removing a hydrogen atom, hydroxy group, etc. from that molecule.

[0081] "Nucleotide sequence" means the base sequence of the nucleotides that make up an oligonucleotide.

[0082] As used herein, "blunt ends" means that the ends of a double-stranded RNA are paired with neither end protruding.

[0083] As used herein, the term "overhang" refers to a protruding portion on one of the ends of a double-stranded RNA. The term "overhang region" refers to the protruding portion. The overhang region can be of any length, but is preferably 1 to 50 nucleotides long, more preferably 1 to 30 nucleotides long, even more preferably 1 to 15 nucleotides long, and most preferably 2 to 6 nucleotides long. Because the protruding portion does not form a double strand in the double-stranded RNA of the present specification, the overhang is also referred to as a single-stranded overhang.

[0084] "Deoxyribonucleotide" refers to a molecule in which the sugar is 2-deoxyribose, a base is bound to the carbon atom at position 1 of the 2-deoxyribose, and a phosphate group is at position 3 or 5. The deoxyribonucleotide of the present invention may be a naturally occurring deoxyribonucleotide, or a deoxyribonucleotide in which the base moiety or the phosphodiester bond moiety of a naturally occurring deoxyribonucleotide has been modified. A single deoxyribonucleotide may be modified by a combination of multiple types of modifications to the base moiety or the phosphodiester bond moiety. The modified deoxyribonucleotides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and RNA, 2023, 29, pp. 423-433.

[0085] When the "deoxyribonucleotides" constitute each strand of oligonucleotide contained in the double-stranded RNA of the present invention, the 3' position of the deoxyribonucleotide is typically linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the deoxyribonucleotide is typically linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The 3'-terminal deoxyribonucleotide of each strand of oligonucleotide contained in the double-stranded RNA of the present invention preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. The 5'-terminal deoxyribonucleotide of each strand of oligonucleotide contained in the double-stranded RNA preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0086] "Ribonucleotide" refers to a molecule in which the sugar is ribose, a base is bound to the carbon atom at position 1 of the ribose, and a phosphate group is located at position 2, 3, or 5. The ribonucleotide of the present invention may be a naturally occurring ribonucleotide, or a naturally occurring ribonucleotide in which the base moiety or the phosphodiester linkage moiety has been modified. A single ribonucleotide may be modified by a combination of multiple types of modifications to the base moiety or the phosphodiester linkage moiety. The modified ribonucleotides are described, for example, in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and RNA, 2023, 29, pp. 423-433.

[0087] When the "ribonucleotides" constitute each strand of oligonucleotide contained in the double-stranded RNA of the present invention, the 3' position of the ribonucleotide is typically linked to another nucleotide via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the ribonucleotide is linked to another nucleotide via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The 3'-terminal ribonucleotide of each strand of oligonucleotide contained in the double-stranded RNA of the present invention preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. The 5'-terminal ribonucleotide of each strand of oligonucleotide contained in the double-stranded RNA preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0088] "Modified sugars" include: (Z1) molecules in which ribose or 2-deoxyribose is partially replaced with one or more substituents, (Z2) pentose or hexose sugars different from ribose and 2-deoxyribose (e.g., hexitol, threose, etc.), and (Z3) molecules in which the entire ribose or 2-deoxyribose, or the tetrahydrofuran ring thereof, is replaced with a 5- to 7-membered saturated or unsaturated ring (e.g., cyclohexane, cyclohexene, morpholine, etc.). Modified sugars include "2-modified sugars," "2-4-linked sugars," and "5-modified sugars," which are described below. Examples of modified sugars and sugar-modified nucleotides described below include sugars and sugar-modified nucleotides disclosed in JP-A-10-304889, WO 2005 / 021570, JP-A-10-195098, JP-T-2002-521310, WO 2007 / 143315, WO 2008 / 043753, WO 2008 / 029619, WO 2008 / 049085, and WO 2017 / 142054. Modified sugars and sugar-modified nucleotides are also disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; RNA, 2023, 29, pp. 423-433; and WO 2018 / 155450.

[0089] Examples of modified sugars that are partially substituted with one substituent include ribose or 2-deoxyribose substituted at any position of the sugar moiety with one or more (preferably one or two) of the following substituents (i) or (ii): (i) a C1-6 alkyl group. When substitutions with two or more C1-6 alkyl groups are contained, the two or more C1-6 alkyl groups may be joined together to form a 3- to 6-membered ring. (ii) a C1-6 alkyl group substituted with at least one selected from the group consisting of a halogen atom, a C1-6 alkoxy group, a haloC1-6 alkoxy group, a mono- or di-C1-6 alkylamino group, a 5- to 10-membered heterocyclic group, a carboxy group, a carbamoyl group, and an N-substituted carbamoyl group. Here, examples of the N-substituted carbamoyl group include an N-methyl-carbamoyl group and an N-ethyl-carbamoyl group, and the methyl group and the ethyl group of the N-methyl-carbamoyl group and the N-ethyl-carbamoyl group are 5- to 10-membered heterocyclic groups or mono- or di-C １－６ It may be substituted with an alkylamino group. Specific examples of the N-substituted carbamoyl group include an N-methylcarbamoyl group, an N-ethylcarbamoyl group, an N-dimethylaminoethyl-carbamoyl group, an N-(morpholinoethyl)carbamoyl group, an N-(2-pyridylethyl)carbamoyl group, and an N-((benzimidazol-1-yl)ethyl)carbamoyl group.

[0090] A "sugar-modified nucleotide" includes not only molecules having the above-mentioned "modified sugar" in place of the sugar moiety of a deoxyribonucleotide or ribonucleotide, but also molecules having an acyclic structure in place of the sugar moiety. For example, sugar-modified nucleotides include "2'-modified nucleotides," "2'-4' bridged nucleotides," "5'-modified nucleotides," and "acyclic nucleotides," which are described below. When the modified sugar is (Z3) as defined above, the sugar-modified nucleotide also includes molecules in which the modified sugar and the nucleobase are linked via a methylene chain or the like.

[0091] "Dimodified sugar" means a non-bridged sugar in which the oxygen atom or carbon atom at position 2 of ribose is modified, and includes "2-O-Me," "2-O-MOE," "2-O-MCE," "2-O-NMA," "2-O-AP," "2-fluoro," "2-O-DMAECE," "2-O-MorECE," "2-O-PyECE," and "2-O-BimECE." Position 4 of a "dimodified sugar" is preferably unmodified. A "2'-modified nucleotide" refers to a molecule having a base bound to the carbon atom at position 1 (position 1 of the ribose before modification) of the "dimodified sugar" and a phosphate group at position 3 or 5, and examples include "2'-O-Me nucleotide," "2'-O-MOE nucleotide," "2'-O-MCE nucleotide," "2'-O-NMA nucleotide," "2'-O-AP nucleotide," "2'-F nucleotide," "2'-O-DMAECE nucleotide," "2'-O-MorECE nucleotide," "2'-O-PyECE nucleotide," and "2'-O-BimECE nucleotide." The 5' position of a 2'-modified nucleotide may or may not be modified.

[0092] "2-O-Me" (also called 2-O-methyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a methoxy group. "2'-O-Me nucleotide" (also called 2'-O-methyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-Me" (position 1 of unmodified ribose) and which has a phosphate group at position 3 or 5.

[0093] "2-O-MOE" (also referred to as 2-O-methoxyethyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a 2-methoxyethyloxy group. "2'-O-MOE nucleotide" (also referred to as 2'-O-methoxyethyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-MOE" (position 1 of the ribose before modification) and which has a phosphate group at position 3 or 5.

[0094] "2-O-MCE" (also called 2-O-methylcarbamoylethyl) refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a methylcarbamoylethyloxy group. "2'-O-MCE nucleotide" (also called 2'-O-methylcarbamoylethyl nucleotide) refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-MCE" (position 1 of the ribose before modification) and which has a phosphate group at position 3 or 5.

[0095] "2-O-NMA" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a [2-(methylamino)-2-oxoethyl]oxy group. "2'-O-NMA nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-NMA" (position 1 of the unmodified ribose) and which has a phosphate group at position 3 or 5.

[0096] "2-O-AP" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a 3-aminopropyloxy group. "2'-O-AP nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-O-AP" (position 1 of the unmodified ribose) and which has a phosphate group at position 3 or 5.

[0097] "2-fluoro" refers to a sugar in which the hydroxy group at position 2 of ribose has been replaced with a fluorine atom. "2'-fluoronucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of "2-fluoro" (position 1 of unmodified ribose) and which has a phosphate group at position 3 or 5.

[0098] "2-O-DMAECE," "2-O-MorECE," "2-O-PyECE," and "2-O-BimECE" are sugars in which the hydroxy group at position 2 of ribose has been replaced with the structures shown below as DMAECE, MorECE, PyECE, and BimECE, respectively. In the structures below, the wavy line indicates the bond position with the carbon atom to which the hydroxy group at position 2 of ribose is bonded.

[0099] "2'-O-DMAECE nucleotide," "2'-O-MorECE nucleotide," "2'-O-PyECE nucleotide," and "2'-O-BimECE nucleotide" refer to molecules in which a base is bound to the carbon atom at position 1 (position 1 of unmodified ribose) of "2-O-DMAECE," "2-O-MorECE," "2-O-PyECE," and "2-O-BimECE," respectively, and which have a phosphate group at position 3 or 5.

[0100] "2'-O-XCE nucleotide" refers to a nucleotide in which the hydroxy group at the 2'-position of a ribonucleotide is modified with a substituted or unsubstituted carbamoylethyl. In some embodiments of the various aspects disclosed herein, the 2'-O-XCE nucleotide is represented by the following formula (I): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group), X is a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a compound represented by the following formula (Ia): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R３ and R ４ each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different}. The wavy line in formula (I) is understood to represent the bonding position to an internucleotide bond with an adjacent nucleotide, the bonding position to a functional molecule or its linker, or the bonding position to a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, and the wavy line in formula (Ia) represents the bonding position to a nitrogen atom in formula (I).

[0101] In some embodiments, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group, wherein the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents, singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group. X is preferably a hydrogen atom or a C1-6 alkyl group, more preferably a C1-3 alkyl group, and even more preferably a methyl group.

[0102] In some embodiments, X is represented by formula (Ia): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted by one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different.

[0103] R １ and R ２ each independently represents a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group. Here, the C1-6 alkyl group and the C2-6 alkenyl group are unsubstituted or substituted with one or more substituents, either singly or differently, selected from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group. Here, when n is 2 or 3, R１ may be the same or different, R ２ may be the same or different. １ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom. ２ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a hydrogen atom.

[0104] Y is NR ３ R ４ When R ３ and R ４ each independently represents a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group, or a C7-10 aralkyl group, wherein the C1-6 alkyl group and C2-6 alkenyl group are unsubstituted or substituted by one or more substituents selected, either singly or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group, and The R group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group. ３ is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a methyl group. ４is preferably a hydrogen atom or a C1-3 alkyl group, more preferably a methyl group. ３ R ４ is N(R ３ ) R ４ and the nitrogen atom may be represented by R ３ and R ４ means that the two are combined.

[0105] In other embodiments, R ３ and R ４ may form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle together with the nitrogen atom to which they are bonded. Here, the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group. R ３ and R ４ However, the 3-11-membered nitrogen-containing non-aromatic heterocycle formed by these together with the nitrogen atom to which they are bonded is preferably a 4-8-membered nitrogen-containing non-aromatic heterocycle containing 4 to 6 methylene groups, such as piperidine, pyrrolidine, morpholine, thiomorpholine, homopiperidine, and homomorpholine. More preferably, it is a ring further containing an oxygen atom or a sulfur atom as a ring-constituting atom, such as morpholine, thiomorpholine, and homomorpholine. Morpholine is particularly preferred. Furthermore, it is preferable that the 4-8-membered nitrogen-containing non-aromatic heterocycle is unsubstituted.

[0106] In another embodiment, Y may be a C2-9 aromatic heterocyclic group, preferably a pyridyl group, an imidazolyl group, or a benzimidazolyl group, more preferably 2-pyridyl, imidazol-1-yl, or (benzimidazol)-1-yl, and particularly preferably 2-pyridyl or (benzimidazol)-1-yl.

[0107] n is the number of repeating structural units and is an integer of 1 to 3, preferably 2.

[0108] The 2'-O-XCE nucleotide may contain the nucleoside structure represented by formula (I) at the 3' or 5' end of the oligonucleotide. When the 2'-O-XCE nucleotide is contained at the 3' end, it may be, for example, the nucleoside structure represented by formula (I-1) below: (X and Base in formula (I-1) have the same meanings as X and Base in formula (I). Z ２ is a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, preferably a hydrogen atom). The wavy line in formula (I-1) is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with the hydrogen atom, the hydroxy-protecting group, or the phosphorus-containing group, etc.

[0109] When the 2'-O-XCE nucleotide is contained at the 5' end, for example, the 2'-O-XCE nucleotide may be represented by the following formula (I-2): (X and Base in formula (I-2) have the same meanings as X and Base in formula (I), and Z １ is a hydroxy group, a phosphate group, or a modified phosphate group). The wavy line in formula (I-2) is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc.

[0110] Exemplary phosphate groups or modified phosphate groups include those compatible with RISC-mediated gene silencing. For example, suitable phosphate groups or modified phosphate groups include monophosphate ((HO) ２ (O)PO-), diphosphoric acid ((HO)２ (O)P-O-P(HO)(O)-O-), triphosphate ((HO) ２ (O)P-O-(HO)(O)P-O-P(HO)(O)-O-), monothiophosphate (phosphorothioate: (HO) ２ (S)P—O—), monodithiophosphate (dithiophosphate: (HO)(HS)(S)P—O—), phosphorothiolate ((HO) ２ (O)P-S-), any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., α-thiotriphosphate, γ-thiotriphosphate, etc.), phosphoramidates ((HO) ２ (O)P-NH-, (HO)(NH ２ ) (O)P—O—), 5′-alkyl phosphonates (alkylene phosphates (e.g., (HO) ２ (O) P-CH ２ -5' (methylene phosphate), (HO) ２ (O) P-CH ２ CH ２ -5'-(ethylene phosphate), R'P(OH)(O)-O-5'- (R'=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-cycloalkylphosphonate ((HO) ２ (O) P-C ３ H ４ -5'-(cyclopropylene phosphate)), 5'-alkenyl phosphonate (alkenyl is, for example, vinyl ((OH) ２ (O)P-CH=CH-5'-), substituted vinyl), 5'-alkoxyalkylphosphonates (R"P(OH)(O)-O-5'- (R"=alkoxyalkyl, for example, methoxymethyl, ethoxymethyl, etc.)).

[0111] In some embodiments, Z １ is preferably a hydroxy group. １ is monophosphate ((HO) ２ (O)P—O—) is preferred.

[0112] In some embodiments, when a 2'-O-XCE nucleotide is included at the 5' end, it is represented by the following formula (II-1): [X and Base in formula (II-1) have the same meanings as X and Base in formula (II), １ is represented by the following formula (IIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], A １ is represented by the following formula (IIc): [Q １ and Q ２ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, and an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group)}. The wavy line in formula (II-1) is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy protecting group, or a phosphorus-containing group, etc., and the wavy line in formula (IIb) represents A in formula (II-1). １ The wavy lines in formula (IIc) indicate the bonding positions of T in formula (II-1), １ The bond positions to the hydroxyl group and the sugar carbon atom are shown.

[0113] In some embodiments, Ra and Rc are preferably hydroxy groups, and Rb is preferably an oxygen atom.

[0114] In some other embodiments, Q １ and Q ２ is preferably a hydrogen atom.

[0115] In some embodiments, when the nucleoside structure represented by formula (I) is contained at the 5' end of an oligonucleotide, it can be represented by the following formula (II-2): (X and Base in formula (II-2) have the same meanings as X and Base in formula (II)) A structure (5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide) is preferred. The wavy line in formula (II-2) is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc.

[0116] In some embodiments, when a 2'-O-XCE nucleotide is included at the 5' end, it is represented by the following formula (III-1): {T in formula (III-1) １ , X and Base are T in formula (III). １ , X and Base; B １ is represented by the following formula (IIIc): [Q ３ and Q ４ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group)}. The wavy lines in formula (III-1) are understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc., and the wavy lines in formula (IIIc) each represent the T １The bond positions to the hydroxyl group and the sugar carbon atom are shown.

[0117] In some other embodiments, Q ３ and Q ４ is preferably a hydrogen atom.

[0118] In some embodiments, when the nucleoside structure represented by formula (I) is contained at the 5' end of an oligonucleotide, it may be represented by the following formula (III-2): (X and Base in formula (III-2) have the same meanings as X and Base in formula (III)) (5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide) is preferred. The wavy lines in formula (III-2) are understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with a functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, and the wavy lines in formula (IIIc) represent the T １ The bond positions to the hydroxyl group and the sugar carbon atom are shown.

[0119] In some embodiments, when a 2'-O-XCE nucleotide is included at the 5' end, it is represented by the following formula (IV-1): {T in formula (IV-1) １ , X and Base are T in formula (IV). １ , X and Base; E １ is represented by the following formula (IVc): [Q ５ ~Q ８are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group)}. The wavy lines in formula (IV-1) are understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, and the wavy lines in formula (IIIc) each represent the T １ The bond positions to the hydroxyl group and the sugar carbon atom are shown.

[0120] In some other embodiments, Q ５ ~Q ８ is preferably a hydrogen atom.

[0121] In some embodiments, when the nucleoside structure represented by formula (I) is contained at the 5' end of an oligonucleotide, it may be represented by the following formula (IV-2): (X and Base in formula (IV-2) have the same meanings as X and Base in formula (IV)) A structure (5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide) is preferred. The wavy line in formula (IV-2) is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with the functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc.

[0122] In some embodiments, the T of formulas (II-1), (III-1) and (IV-1) １In the formulas (II-1), (III-1) and (IV-1), Rb is preferably an oxygen atom, and Ra and Rc are each independently preferably a hydroxy group or a protected hydroxy group, more preferably a protected hydroxy group, even more preferably an acyloxymethyl group (e.g., a methyl group substituted with a C1-6 alkylcarbonyloxy group) or an alkyl group (e.g., a C1-6 alkyl group), and particularly preferably a pivaloyloxymethyl group or an ethyl group. The preferred structures of X in the formulas (II-1), (III-1) and (IV-1) are the same as the preferred corresponding structures encompassed in the double-stranded RNA agents. Z in the formulas (II-1), (III-1) and (IV-1) ３ The hydroxy-protecting group in is preferably a silyl group, and particularly preferably a tert-butyldimethylsilyl group.

[0123] The compounds represented by formula (I) to formula (IV-2) may have isomers. In such cases, the compounds of the present embodiment are not limited to a specific isomer, and include all possible isomers (e.g., keto-enol isomers, imine-enamine isomers, diastereoisomers, optical isomers, rotamers, etc.), racemates, and mixtures thereof.

[0124] The "2'-modified nucleotide" includes a "2'-O-XCE nucleotide", and the "2'-O-R-XCE nucleotide" includes a "2'-O-MCE nucleotide", a "2'-O-DMAECE nucleotide", a "2'-O-MorECE nucleotide", a "2'-O-PyECE nucleotide", and a "2'-O-BimECE nucleotide".

[0125] A "2-4 bridged sugar" refers to a sugar in which the bridge unit is substituted at two positions, the 2- and 4-positions of ribose. Examples of the bridge unit include a C2-6 alkylene group (the alkylene group is unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen atom, an oxo group, and a thioxo group, and one or two methylene groups of the alkylene group are unsubstituted or independently substituted with a group selected from the group consisting of -O-, -NR1- (R1 is a hydrogen atom, a C1-6 alkyl group, or a halo-C1-6 alkyl group), and -S-). The 5-position of a "2-4 bridged sugar" may or may not be modified, but is preferably unmodified.

[0126] The term "2',4' bridged nucleotide" (2',4'-BNA) refers to a molecule in which a base is bound to the carbon atom at position 1 (position 1 of unmodified ribose or 2-deoxyribose) of the "2,4-bridged sugar" and a nucleic acid base is attached to a phosphate group at position 3 or 5. For example, β-D-methyleneoxy (4'-CH), also known as LNA (Locked Nucleic Acid (registered trademark)), is used. ２ -O-2')BNA or α-L-methyleneoxy(4'-CH ２ -O-2')BNA, also known as ENA, ２ ) ２ -O-2')BNA, (4β-D-thio'-CH ２ -S-2')BNA, aminooxy (4'-CH ２ -O-N(R １１ )-2')BNA(R １１ is a hydrogen atom or methyl), oxyamino (4'-CH ２ -N(R １２ )-O-2')BNA(R １２ is a hydrogen atom or methyl), 2',4'-BNACOC, 3'-amino-2',4'-BNA, 5'-methylBNA, also called cEt (4'-CH(CH ３ )-O-2')BNA, also called cMOE-BNA (4'-CH(CH ２ OCH ３)-O-2') BNA, amide type BNA (also called AmNA) (4'-C(=O)-N(R １３ )-2')BNA(R １３ is a hydrogen atom or methyl), also called scpBNA (4'-C(spiro-cyclopropyl)-O-2')BNA, also called GuNA (4'-CH ２ -N(R １４ )-2')BNA(R １４ is C(=NHR １５ +) NHR １６ and R １５ , R １６ are each independently a hydrogen atom, methyl, ethyl, isopropyl, or t-butyl), also known as amino LNA (4'-CH ２ -N(R １７ )-2')BNA(R １７ is a hydrogen atom or methyl), other BNAs known to those skilled in the art, etc.

[0127] A "5-modified sugar" refers to an unbridged sugar in which the oxygen or carbon atom at position 5 of the ribose backbone has been modified, and includes "5-CP," "5-methyl," and "5-aminopropyl." Positions 2 and 4 of a "5-modified sugar" are preferably unmodified. A "5'-modified nucleotide" refers to a molecule in which a base is bound to the carbon atom at position 1 of the "5-modified sugar" (position 1 of the 2-deoxyribose before modification) and a nucleic acid base is attached to a phosphate group at position 2, 3, or 5, and includes, for example, a "5'-CP nucleotide," a "5'-methyl nucleotide," and a "5'-aminopropyl nucleotide." The 2' position of a 5'-modified nucleotide may or may not be modified. The 2' and 4' positions of a 5'-modified nucleotide may or may not be bridged, but are preferably unbridged.

[0128] "5-CP" is a sugar in which the 5-position of the ribose backbone is substituted with two methyl groups, and these two methyl groups together form a cyclopropane. A "5'-CP nucleotide" is a molecule in which the sugar is the above-mentioned "5-CP," a base is bound to the carbon atom at position 1 (position 1 of the base 2-deoxyribose), and a phosphate group is bound to position 3 or 5, and can be represented by the following structural formula. In the formula, Base is a nucleic acid base. The wavy line is understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with a functional molecule or its linker, or the bonding position with a hydrogen atom, a hydroxy protecting group, or a phosphorus-containing group, etc. "5-methyl" is a sugar in which the 5-position of the ribose backbone is substituted with a methyl group. "5-aminopropyl" is a sugar in which the 5-position of the ribose backbone is substituted with a 3-aminopropyl group. "5'-methyl nucleotide" and "5'-aminopropyl nucleotide" refer to molecules in which a base is bound to the carbon atom at position 1 of "5-methyl" and "5-aminopropyl" (position 1 of the base 2-deoxyribose) and a phosphate group is at position 3 or 5, respectively.

[0129] "5-vinyl" is a sugar in which the methylene group at the 5-position of ribose is replaced with an ethene-1,2-diyl group (vinyl group). "5-CP" is a sugar in which the methylene group at the 5-position of ribose is replaced with a cyclopropane-1,1-diyl group (cyclopropyl group).

[0130] Vinylphosphonate (VP) nucleotides, cyclopropanephosphonate (CPP) nucleotides, and ethylphosphonate (EP) nucleotides, which will be described later, are also included in the 5'-modified nucleotides.

[0131] In the "deoxyribonucleotide," "ribonucleotide," "2'-modified nucleotide," "2'-4' bridged nucleotide," and "5'-modified nucleotide," the bond between the carbon atom at the 1' position and the base can be an α-glycosidic bond or a β-glycosidic bond, but is usually a β-glycosidic bond. Therefore, β-D-methyleneoxy BNA is usually used as LNA.

[0132] When the "sugar-modified nucleotide" constitutes each strand of oligonucleotide contained in the double-stranded RNA of the present invention, for example, the 3' position of the sugar-modified nucleotide is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the sugar-modified nucleotide is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The sugar-modified nucleotide at the 3' end of each strand of oligonucleotide contained in the double-stranded RNA of the present invention preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. The sugar-modified nucleotide at the 5' end of each strand of oligonucleotide contained in the double-stranded RNA preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0133] Examples of modifications of the phosphodiester bond in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides include phosphorothioation, methylphosphonation (including chiral-methylphosphonation), methylthiophosphonation, phosphorodithioation, phosphoramidate, phosphorodiamidate, phosphoramidothioate, boranophosphate, phosphorylguanidination, mesylamidation, and the like. Furthermore, examples of modifications of phosphodiester bond moieties in nucleotides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; RNA, 2023, 29, pp. 423-433, etc., and these can be used for the phosphodiester bond moieties in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides.

[0134] A "bridged nucleotide" is a sugar-modified nucleotide in which the bridge unit is substituted by two substitutions in the sugar moiety, and examples thereof include 2'-4' bridged nucleotides.

[0135] "Acyclic nucleotide" refers to any nucleotide that has an acyclic structure in place of the sugar of a ribonucleotide or deoxyribonucleotide. For example, in an acyclic nucleotide, any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') are absent, and / or at least one of the ribose carbon atoms or oxygen atoms (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is absent from the nucleotide. In some embodiments, an acyclic nucleotide has the following formula (V): (Base in formula (V) has the same meaning as Base in formula (I), and R ３６ and R ３７ are independently a hydrogen atom, a halogen atom, a hydroxy group, a C1-6 alkoxy group, or a C1-6 alkyl group.

[0136] "UNA" is referred to as an unlocked acyclic nucleic acid and is a type of acyclic nucleotide herein. In a UNA, either the sugar bond of a ribonucleotide or a deoxyribonucleotide has been removed to form an unlocked "sugar" residue. In one example, a UNA includes a monomer in which the C1'-C4' bond (i.e., the covalent carbon-oxygen-carbon bond between the C1' and C4' carbons) has been removed. In another example, a UNA includes a monomer in which the C2'-C3' bond of the sugar (i.e., the covalent carbon-carbon bond between the C2' and C3' carbons) can be removed (see Tetrahedron Letters, 1985, 26(17), p. 2059 and Mol. Biosyst., 2009, 10, p. 1039, which are hereby incorporated by reference in their entireties). The acyclic structure allows for backbone flexibility without affecting Watson-Crick pairing. The UNA can be linked by a 2'-5' or a 3'-5' linkage.

[0137] "GNA" is commonly referred to as glycol nucleic acid and is a type of acyclic nucleotide herein. GNA is composed of repeating units of glycerol linked by phosphodiester or modified phosphodiester bonds. It is similar to DNA or RNA (deoxyribonucleotides or ribonucleotides) although the "backbone" composition is different. Its representative structure is shown in the following formula (VI): (Base in formula (VI) has the same meaning as Base in formula (I)).

[0138] Sugar-modified nucleotides are not limited to those exemplified herein. Many sugar-modified nucleotides are known in the art, and sugar-modified nucleotides described in, for example, U.S. Patent No. 8,299,039 to Tachas et al. (especially columns 17 to 22), Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667, Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471, Future Medicinal Chemistry, 2011, 3, pp. 339-365, etc., can also be used in embodiments of the present invention.

[0139] "Nucleobase" generally refers to a base component constituting a nucleic acid, and natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Natural nucleobases and modified nucleobases thereof can be used in the base moiety of deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides used herein. Modified nucleobases can form base pairs (i.e., hydrogen bonds) with any nucleobase (preferably a base complementary to the nucleobase before modification). Typically, modified nucleobases include 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and These include thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils, and cytosine, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is substituted with other heterocycles, such as 7-deazaadenine, 7-deazaguanosine, 2-aminopyridine, and 2-pyridone. Examples of modifications of the base moiety in nucleotides are disclosed in Journal of Medicinal Chemistry, 2016, 59, pp. 9645-9667; Medicinal Chemistry Communication, 2014, 5, pp. 1454-1471; Future Medicinal Chemistry, 2011, 3, pp. 339-365; and International Publication No. WO 2007 / 090071, among others. These modifications can be used for the base moiety in deoxyribonucleotides, ribonucleotides, and sugar-modified nucleotides. The amino and hydroxy groups in the base moiety may each be independently protected.

[0140] The base moiety in the deoxyribonucleotide, ribonucleotide, and sugar-modified nucleotide is preferably at least one selected from the group consisting of adenine (A), guanine (G), thymine (T), cytosine (C), uracil (U), and 5-methylcytosine (5-me-C).

[0141] A "thermo-destabilizing modification" is a modification to a double-stranded RNA that results in a double-stranded RNA with an overall lower melting temperature (Tm) (preferably 1, 2, 3 to 4 degrees lower) than the Tm of a double-stranded RNA that does not have the one or more modifications. "Thermo-destabilizing modifications" include, but are not limited to, abasic modifications, mismatches with opposite nucleotides in the opposite strand, 2'-deoxy modifications, acyclic modifications (e.g., unlocked nucleic acids (UNA) or glycol nucleic acids (GNA)), and the like. A "thermo-destabilizing sugar modification" refers to a thermo-destabilizing modification that uses a sugar-modified nucleotide, and may include the 2'-deoxy modifications, acyclic modifications, sugar modifications, and the like.

[0142] An "abasic nucleotide" is a nucleotide that does not have a nucleobase. Abasic modification can be achieved by using an abasic nucleotide. Exemplary nucleoside structures used as abasic modifications include, but are not limited to, the following formula (VII): (In formula (VII), R ３１ is a hydrogen atom, a methyl group, an ethyl group, or a methoxy group, and R ３２ is a hydrogen atom, a methyl group, an ethyl group, or a methoxy group, and R ３３ represents a hydrogen atom, a methyl group, an ethyl group, or a methoxy group, and * represents that the stereocenter is either R or S, or a racemic mixture.

[0143] Exemplary nucleoside structures for use as heat-labile sugar modifications include, but are not limited to, those of the following formula (VIII): (Base in formula (VIII) has the same meaning as Base in formula (I), and R ３４ is a fluorine or methoxy group, and * indicates that the stereocenter is either R or S, or a racemic mixture).

[0144] Nucleoside structures used as additional heat-labile sugar modifications include, but are not limited to, those of the following formula (IX): (Base in formula (IX) has the same meaning as Base in formula (I), and R ３５ is a hydrogen atom, a hydroxy group, or a C1-6 alkoxy group).

[0145] In some embodiments, nucleoside structures used as thermolabile sugar modifications include those of the following formula (X): (Base in formula (X) has the same meaning as Base in formula (I), and * represents a racemate or an R or S stereocenter.) The wavy lines in formulas (V) to (X) are understood to represent the bonding position of an internucleotide bond with an adjacent nucleotide, the bonding position with a functional molecule or its linker, the bonding position with a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc.

[0146] The thermally destabilizing modification of double-stranded RNA molecules can be mismatched (i.e., non-complementary base pairing) between the thermally destabilizing nucleotide in the dsRNA duplex and the opposite nucleotide of the opposite strand.Exemplary mismatched base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or combinations thereof.Other mismatched base pairings known in the art are also suitable for the present invention.Mismatches can occur between any nucleotide, whether naturally occurring or modified, i.e., mismatched base pairings can occur between the nucleobases from each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.

[0147] In some embodiments, the thermodestabilizing modifications in the seed region of the antisense strand include nucleotides that exhibit impaired Watson-Crick bonding with complementary bases on the target RNA. Exemplary nucleotides that exhibit impaired Watson-Crick bonding with complementary bases on the target RNA include, but are not limited to, nucleotides having the following formula (XI): Nucleotides containing nucleobases independently selected from:

[0148] Further examples of abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0149] Thermodestabilizing modifications can also include universal nucleobases and phosphodiester bond modifications that reduce or eliminate their ability to form hydrogen bonds with opposing bases.

[0150] In some embodiments, thermal destabilizing modifications include but are not limited to the nucleotides with non-standard bases, such as the nucleobase modifications that impair or completely eliminate the ability to form hydrogen bonds with the base of the opposite strand.These nucleobase modifications have been evaluated for destabilizing the central region of dsRNA duplex, as described in International Publication No. 2010 / 0011895 (which is incorporated herein by reference in its entirety).Exemplary of such nucleobase modifications are as follows: In this way, some of the thermally destabilizing modifications can be included in the modified nucleobase. Note that the wavy lines in formulas (XI) to (XII) are understood to represent the bonding positions of the sugar carbon atoms of the nucleotides, etc.

[0151] In some embodiments, the compound of formula (XIII) below: (In the formula, R ３８ is a hydrogen atom, a hydroxy group, a C1-6 alkoxy group, fluorine, an amino group, an N-methylamino group, or an N,N'-dimethylamino group). The wavy line in formula (XIII) is understood to represent the bonding position to the internucleotide bond with the adjacent nucleotide, the bonding position to the functional molecule or its linker, the bonding position to a hydrogen atom, a hydroxy protecting group, or a phosphorus-containing group, etc.

[0152] Exemplary phosphodiester bond modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester bonds include, but are not limited to, the following formula (XIV): (In the formula, R ３９is a C1-6 alkyl group, and examples of C1-6 alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl. The wavy lines in formula (XIV) are understood to represent the bonding positions of the sugar carbon atoms of the nucleotide, etc. Thus, some of the heat-labile modifications may be included in the modified phosphodiester bond.

[0153] In some embodiments, the thermolabilizing modification has the following formula (XV): (Base in formula (XV) has the same meaning as Base in formula (I), and R ３４ is R in formula (VIII). ３４ The wavy line in formula (XV) is understood to represent the bonding position to an internucleotide bond between adjacent nucleotides, the bonding position to a functional molecule or a linker thereof, or the bonding position to a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, etc.

[0154] When the "thermally destabilizing modification" constitutes each strand of oligonucleotide contained in the double-stranded RNA of the present invention, for example, the 3' position of the thermolabile modification is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond), and the 5' position of the thermolabile modification is linked to another nucleotide or the like via a phosphodiester bond or a modified phosphodiester bond (e.g., a phosphorothioate bond). The thermolabile modification at the 3' end of each strand of oligonucleotide contained in the double-stranded RNA of the present invention preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 3' position, and the 5' position is as described above. The thermolabile modification at the 5' end of each strand of oligonucleotide contained in the double-stranded RNA, for example, preferably has a hydroxy group, a phosphate group, or a modified phosphate group at its 5' position, and the 3' position is as described above.

[0155] Some of the "thermally destabilizing modifications" may be included in the "sugar-modified nucleotides."

[0156] Next, the double-stranded RNA of the present invention will be described. Hereinafter, in this specification, double-stranded RNA may be referred to as "dsRNA."

[0157] In one aspect, the present invention provides a dsRNA, the dsRNA being capable of inhibiting expression of a target RNA, the dsRNA comprising a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand), wherein the antisense strand has sufficient complementarity with a target sequence to mediate RNA interference, and the dsRNA comprises at least one 2'-O-XCE nucleotide.

[0158] Each strand of the dsRNA independently has a length of 14 to 40 nucleotides. For example, each strand independently has a length of 14 to 40, 17 to 37, 25 to 37, 27 to 30, 17 to 23, 17 to 21, 17 to 19, 19 to 25, 19 to 23, 19 to 21, 21 to 25, or 21 to 23 nucleotides. Without limitation, the sense and antisense strands can be of equal or unequal length. In some embodiments, the antisense strand is longer than the sense strand, for example, by 1, 2, 3, 4, or 5 nucleotides.

[0159] In some embodiments, the antisense strand is 18-35 nucleotides in length. In some embodiments, the antisense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In certain embodiments, the antisense strand is 23 nucleotides in length. In certain embodiments, the antisense strand is 22 nucleotides in length. In other certain embodiments, the antisense strand is 21 nucleotides in length.

[0160] Like the antisense strand, the sense strand, in some embodiments, is 18-35 nucleotides in length. In some embodiments, the sense strand is 21-25, 19-25, 19-21, or 21-23 nucleotides in length. In some particular embodiments, the sense strand is 21 nucleotides in length. In some particular embodiments, the sense strand is 20 nucleotides in length. In some particular embodiments, the sense strand is 19 nucleotides in length.

[0161] In certain embodiments, the sense strand is 21 nucleotides long and the antisense strand is 23 nucleotides long. In certain embodiments, the sense strand is 20 nucleotides long and the antisense strand is 23 nucleotides long. In certain embodiments, the sense strand is 19 nucleotides long and the antisense strand is 23 nucleotides long. In certain embodiments, the sense strand is 20 nucleotides long and the antisense strand is 22 nucleotides long. In certain embodiments, the sense strand is 21 nucleotides long and the antisense strand is 21 nucleotides long. In certain embodiments, the sense strand is 19 nucleotides long and the antisense strand is 21 nucleotides long.

[0162] The double-stranded RNA has a hybridizing portion. Generally, the hybridizing portion is 12 to 40 nucleotide base pairs in length. For example, the dsRNA has a hybridizing portion that is 12 to 25 nucleotide base pairs in length. In some embodiments, the dsRNA has a hybridizing portion that is 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In certain embodiments, the dsRNA has a hybridizing portion that is 19, 20, 21, or 22 nucleotide base pairs in length. In even more certain embodiments, the dsRNA has a hybridizing portion that is 21 nucleotide base pairs in length.

[0163] The 2'-O-XCE nucleotide can be present anywhere within the dsRNA. For example, the 2'-O-XCE nucleotide can be present in the sense strand of the dsRNA. In general, the 2'-O-XCE nucleotide can be present anywhere within the antisense strand. For example, the 2'-O-XCE nucleotide can be at the 5' end of the sense strand, the 3' end of the sense strand, and / or at an internal position within the sense strand.

[0164] In some embodiments, the 2'-O-XCE nucleotide is present at the 5'-end of the sense strand, hi some embodiments, the 2'-O-XCE nucleotide is present at the 3'-end of the sense strand.

[0165] In some embodiments, the 2'-O-XCE nucleotide can be present at an internal position in the sense strand. For example, the 2'-O-XCE nucleotide can be at position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, counting from the 5' end of the sense strand. In some embodiments, the 2'-O-XCE nucleotide can be present within the 5' region (i.e., positions 2-8, counting from the 5' end of the sense strand). In some embodiments, the 2'-O-XCE nucleotide can be present in the central region of the sense strand.

[0166] In some embodiments, the 2'-O-XCE nucleotide may be present in the sense strand at a position opposite the seed region of the antisense strand (ie, at positions 2 to 8 counting from the 5' end of the hybridizing portion of the antisense strand).

[0167] In some embodiments, the sense strand comprises at least one 2'-O-XCE nucleotide, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more. In some embodiments, the sense strand comprises at least one 2'-O-XCE nucleotide. In some embodiments, the sense strand preferably comprises at least one 2'-O-XCE nucleotide at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises 1 to 10 (more preferably 2 to 10, even more preferably 2 to 4, and even more preferably 2 or 4) 2'-O-XCE nucleotides. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1, 2, 20, and 21, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1 and 21, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises at least two 2'-O-XCE nucleotides. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at at least two of the following positions, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at at least two of the following positions, 1, 2, 20, and 21, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 21, counting from the 3' end of the sense strand hybridizing portion.In some embodiments, the sense strand comprises at least three 2'-O-XCE nucleotides. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at at least three positions: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at at least three positions: 1, 2, 20, and 21, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises at least four 2'-O-XCE nucleotides. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at at least four of the following positions, counting from the 3' end of the sense strand hybridizing portion: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, and 21. In some embodiments, the sense strand preferably comprises 2'-O-XCE nucleotides at positions 1, 2, 20, and 21, counting from the 3' end of the sense strand hybridizing portion.

[0168] In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1 and 2 counting from the 5'-end of the sense strand, more preferably at positions 1 and 2 counting from the 5'-end of the sense strand. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1 and 2 counting from the 3'-end of the sense strand, more preferably at positions 1 and 2 counting from the 3'-end of the sense strand. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1 and 2 counting from the 5'-end of the sense strand and positions 1 and 2 counting from the 3'-end of the sense strand, more preferably at all of positions 1 and 2 counting from the 5'-end of the sense strand and positions 1 and 2 counting from the 3'-end of the sense strand.

[0169] In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the sense strand. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at position 1, counting from the 3'-end of the sense strand. In some embodiments, the sense strand preferably comprises a 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the sense strand and at position 1, counting from the 3'-end of the sense strand.

[0170] In some embodiments, the sense strand comprises at least two or more 2'-O-XCE nucleotides. When two or more 2'-O-XCE nucleotides are present, they can be adjacent to one another. Thus, in some embodiments, the sense strand comprises at least two, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more 2'-O-XCE nucleotides that are adjacent to one another.

[0171] In some embodiments, the sense strand comprises three 2'-O-XCE nucleotides that are adjacent to each other.

[0172] In some embodiments, the 2'-O-XCE nucleotide is present in the antisense strand. Generally, the 2'-O-XCE nucleotide can be present anywhere within the antisense strand. For example, the 2'-O-XCE nucleotide can be at the 5' end of the antisense strand, the 3' end of the antisense strand, and / or at an internal position within the antisense strand.

[0173] In some embodiments, the 2'-O-XCE nucleotides are present at the 5'-end of the antisense strand. In some embodiments, the 2'-O-XCE nucleotides are present at the 3'-end of the antisense strand.

[0174] In some embodiments, the 2'-O-XCE nucleotide may be located at an internal position in the antisense strand. For example, the 2'-O-XCE nucleotide may be located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the 2'-O-XCE nucleotide may be located within the seed region (i.e., positions 2 to 8, counting from the 5' end of the antisense strand hybridizing portion). Positions 2 to 8, counting from the 5' end of the antisense strand hybridizing portion, are referred to as the seed region. The seed region is responsible for initial recognition of the target RNA. Introducing a thermolabile modification into the seed region can reduce off-target effects. For example, the antisense strand comprises a 2'-O-XCE nucleotide at at least one of positions 3 to 8, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the 2'-O-XCE nucleotide can be present in the central region of the antisense strand.

[0175] In some embodiments, the antisense strand comprises at least 1, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or more 2'-O-XCE nucleotides. In some embodiments, the antisense strand comprises at least one 2'-O-XCE nucleotide. In some embodiments, the antisense strand comprises 1-10 (preferably 1-9, more preferably 1, 2, 3, or 4) 2'-O-XCE nucleotides. In some embodiments, the antisense strand preferably comprises at least one 2'-O-XCE nucleotide at positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises a 2'-O-XCE nucleotide at at least one of positions 1, 5, and 10, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises at least two 2'-O-XCE nucleotides. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at at least two positions: 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at at least two positions: 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at at least two positions: 1, 5, and 10, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 3, counting from the 5' end of the antisense strand hybridizing portion.In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 4, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 5, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 6, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 7, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 8, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 9, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 1 and 10, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at positions 5 and 10, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand preferably comprises at least three 2'-O-XCE nucleotides. In some embodiments, the antisense strand preferably comprises 2'-O-XCE nucleotides at at least three of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, and 23, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, it is more preferred that the antisense strand comprises 2'-O-XCE nucleotides at at least three of the following positions, 1, 3, 4, 5, 6, 7, 8, 9, and 10, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, it is especially preferred that the antisense strand comprises 2'-O-XCE nucleotides at positions 1, 5, and 10, counting from the 5' end of the antisense strand hybridizing portion.

[0176] In some embodiments, the antisense strand comprises at least two or more 2'-O-XCE nucleotides. When two or more 2'-O-XCE nucleotides are present, they may be adjacent to one another. Thus, in some embodiments, the antisense strand comprises at least two, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, 2'-O-XCE nucleotides that are adjacent to one another.

[0177] In some embodiments, the antisense strand comprises three 2'-O-XCE nucleotides adjacent to each other.

[0178] In some embodiments, the 2'-O-XCE nucleotide is a 2'-O-MCE.

[0179] In some embodiments, the 2'-O-XCE nucleotide is 2'-O-DMAECE.

[0180] In some embodiments, the 2'-O-XCE nucleotide is 2'-O-MorECE.

[0181] In some embodiments, the 2'-O-XCE nucleotide is 2'-O-PyECE.

[0182] In some embodiments, the 2'-O-XCE nucleotide is 2'-O-BimECE.

[0183] When a dsRNA contains at least two or more 2'-O-XCE nucleotides, the 2'-O-XCE nucleotides may be the same or different.

[0184] The double-stranded RNA described herein may contain one or more sugar-modified nucleotides. It is noted that in a double-stranded RNA, one or more sugar-modified nucleotides are present in addition to one or more 2'-O-XCE nucleotides.

[0185] In some embodiments, a dsRNA can include 2'-fluoro nucleotides. For example, a dsRNA can include at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more, 2'-fluoro nucleotides. Without limitation, the 2'-fluoro nucleotides can all be present in one strand. In some embodiments, both the sense strand and the antisense strand include at least two 2'-fluoro nucleotides. The 2'-fluoro nucleotides can appear at any nucleotide in the sense strand or the antisense strand. For example, 2'-fluoro nucleotides can appear at every nucleotide on the sense strand and / or the antisense strand; each 2'-fluoro nucleotide can appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand include 2'-fluoro nucleotides in an alternating pattern. The alternating pattern of 2'-fluoro nucleotides on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-fluoro nucleotides on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro nucleotides on the antisense strand.

[0186] The antisense strand of a dsRNA may comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-fluoro nucleotide. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, or 6 2'-fluoro nucleotides. Without limitation, the 2'-fluoro nucleotides in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises at least one 2'-fluoro nucleotide. For example, the antisense strand comprises a 2'-fluoro nucleotide at at least one position selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises a 2'-fluoro nucleotide at at least one position selected from positions 2 or 14, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at at least two positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand comprises at least three 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at at least three positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand comprises at least four 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at at least four positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. Preferably, the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion.In some other embodiments, the antisense strand comprises at least five 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at at least five positions selected from positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand comprises at least six 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion.

[0187] The sense strand of a dsRNA can comprise at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 2'-fluoro nucleotide. In some embodiments, the sense strand comprises 1, 2, 3, 4, or 5 2'-fluoro nucleotides. Without limitation, the 2'-fluoro nucleotides in the sense strand can be located at any position. In some embodiments, the sense strand comprises at least one 2'-fluoro nucleotide. For example, the sense strand comprises a 2'-fluoro nucleotide at one position selected from 11, 12, 13, or 15, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises at least two 2'-fluoro nucleotides. For example, the sense strand comprises a 2'-fluoro nucleotide at two positions selected from 11, 12, 13, or 15, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises at least three 2'-fluoro nucleotides. For example, the sense strand comprises 2'-fluoro nucleotides at three positions selected from positions 11, 12, 13, or 15, counting from the 3' end of the sense strand hybridizing portion. In some other embodiments, the sense strand comprises at least four 2'-fluoro nucleotides. For example, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion.

[0188] In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at at least one position opposite or complementary to position 11, 12, or 13, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises a 2'-fluoro nucleotide at at least one position opposite or complementary to position 11, 12, or 15, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises a 2'-fluoro nucleotide at at least one position opposite or complementary to position 11, 12, 13, or 15, counting from the 5' end of the antisense strand hybridizing portion.

[0189] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11 and 12, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11 and 13, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11 and 13, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In yet some other embodiments, the sense strand comprises 2'-fluoro nucleotides at least at positions 11, 13, and 15 from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end of the antisense strand hybridizing portion.

[0190] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion.In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In yet some other embodiments, the sense strand contains 2'-fluoro nucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion.

[0191] In some embodiments, the antisense strand does not contain 2'-fluoro nucleotides at positions 3-9, counting from the 5' end of the antisense strand hybridizing portion.

[0192] A dsRNA can comprise at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more 2'-O-Me nucleotides. Without limitation, the 2'-O-Me nucleotides can all be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least one 2'-O-Me nucleotide. The 2'-O-Me nucleotide can appear at any nucleotide in the sense strand or the antisense strand. For example, the 2'-O-Me nucleotide can appear at every nucleotide on the sense strand and / or the antisense strand; the 2'-O-Me nucleotides can appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand comprise 2'-O-Me nucleotides in an alternating pattern. The alternating pattern of 2'-O-Me nucleotides on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-O-Me nucleotides on the sense strand can be shifted relative to the alternating pattern of 2'-O-Me nucleotides on the antisense strand.

[0193] The antisense strand of the dsRNA can comprise at least 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more 2'-O-Me nucleotides. Without limitation, the 2'-O-Me nucleotides in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises at least 3 2'-O-Me nucleotides.

[0194] In some embodiments, the antisense strand does not contain 2'-O-Me nucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. For example, the antisense strand does not contain 2'-O-Me nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand does not contain 2'-O-Me nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand does not contain 2'-O-Me nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In still some other embodiments, the antisense strand does not contain 2'-O-Me nucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion.

[0195] The sense strand of a dsRNA can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more 2'-O-Me nucleotides. Without limitation, the 2'-O-Me nucleotides in the sense strand can be located at any position. In some embodiments, the sense strand does not comprise a 2'-O-Me nucleotide at positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion. In some other embodiments, the sense strand does not comprise a 2'-O-Me nucleotide at positions 11, 12, and 15, counting from the 3' end of the sense strand hybridizing portion. In some other embodiments, the sense strand does not comprise a 2'-O-Me nucleotide at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises a 2'-O-Me nucleotide at at least one selected from positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises a 2'-O-Me nucleotide at position 11, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises a 2'-O-Me nucleotide at position 12, counting from the 3' end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises a 2'-O-Me nucleotide at position 13, counting from the 3' end of the sense strand hybridizing portion.

[0196] A dsRNA may additionally comprise 2'-modified nucleotides excluding 2'-O-XCE nucleotides, 2'-fluoro nucleotides, and 2'-O-Me nucleotides. For example, a dsRNA may comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more 2'-modified nucleotides. Without limitation, the 2'-modified nucleotides may all be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least 2'-modified nucleotides. The 2'-modified nucleotides may appear at any nucleotide in the sense strand or the antisense strand. For example, the 2'-modified nucleotides may appear at every nucleotide in the sense strand and / or the antisense strand; each 2'-modified nucleotide may appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand comprise 2'-modified nucleotides in an alternating pattern. The alternating pattern of 2'-modified nucleotides on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-modified nucleotides on the sense strand can be shifted relative to the alternating pattern of 2'-modified nucleotides on the antisense strand.

[0197] The antisense strand of the dsRNA can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-modified nucleotides. Without limitation, the 2'-modified nucleotides of the antisense strand can be located at any position.

[0198] The sense strand of a dsRNA can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-modified nucleotides. Without limitation, the 2'-modified nucleotides of the sense strand can be located at any position. In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-modified nucleotides, and the antisense strand does not comprise a 2'-fluoro nucleotide at positions 3 to 9, counting from the 5' end of the antisense strand hybridizing portion.

[0199] A dsRNA can comprise 2'-4' bridged nucleotides. For example, a dsRNA can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more, 2'-4' bridged nucleotides. Without limitation, the 2'-4' bridged nucleotides can all be present in one strand. In some embodiments, both the sense strand and the antisense strand comprise at least a 2'-4' bridged nucleotide. The 2'-4' bridged nucleotide can appear at any nucleotide in the sense strand or the antisense strand. For example, a 2'-4' bridged nucleotide can appear at every nucleotide on the sense strand and / or the antisense strand; each 2'-4' bridged nucleotide can appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand comprise 2'-4' bridged nucleotides in an alternating pattern. The alternating pattern of 2'-4' bridged nucleotides on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of 2'-4' bridged nucleotides on the sense strand can be shifted relative to the alternating pattern of 2'-4' bridged nucleotides on the antisense strand.

[0200] The antisense strand of the dsRNA can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more 2'-4' bridged nucleotides. Without limitation, the 2'-4' bridged nucleotides of the antisense strand can be located in any position.

[0201] The sense strand of a dsRNA can comprise at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, 2'-4' bridged nucleotides. Without limitation, the 2'-4' bridged nucleotides in the sense strand can be located at any position. In some embodiments, the sense strand comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, 2'-4' bridged nucleotides, and the antisense strand does not comprise a 2'-fluoro nucleotide at positions 3 through 9, counting from the 5' end of the antisense strand hybridizing portion.

[0202] In some embodiments, dsRNA can contain deoxyribonucleotides.For example, dsRNA can contain at least 1, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deoxyribonucleotides.Without limitation, all deoxyribonucleotides can be present in one strand.In some embodiments, both the sense strand and the antisense strand contain at least two deoxyribonucleotides.Deoxyribonucleotides can appear at any nucleotide in the sense strand or the antisense strand.For example, deoxyribonucleotides can appear at every nucleotide in the sense strand and / or the antisense strand; each deoxyribonucleotide can appear in an alternating pattern on the sense strand or the antisense strand; or both the sense strand and the antisense strand contain deoxyribonucleotides in an alternating pattern. The alternating pattern of deoxyribonucleotides on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of deoxyribonucleotides on the sense strand can be shifted relative to the alternating pattern of deoxyribonucleotides on the antisense strand.

[0203] The antisense strand of a dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) deoxyribonucleotide. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, or 6 deoxyribonucleotides. Without limitation, the deoxyribonucleotides in the antisense strand may be located at any position. In some embodiments, the antisense strand contains at least one deoxyribonucleotide. For example, the antisense strand contains a deoxyribonucleotide at at least one (e.g., 1 to 6, preferably 2 to 6, 3 to 5) position selected from 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5'-end of the hybridizing portion of the antisense strand. Preferably, the antisense strand comprises a deoxyribonucleotide at at least one (e.g., 1 to 6, preferably 2 to 6, 3 to 5) position selected from 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand comprises at least two deoxyribonucleotides. For example, the antisense strand comprises deoxyribonucleotides at at least two positions selected from 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises deoxyribonucleotides at at least two positions selected from 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some embodiments, the antisense strand comprises at least three deoxyribonucleotides. For example, the antisense strand comprises deoxyribonucleotides at at least three positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises deoxyribonucleotides at at least three positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand comprises at least four deoxyribonucleotides.For example, the antisense strand comprises deoxyribonucleotides at at least four positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises deoxyribonucleotides at at least four positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand comprises at least five deoxyribonucleotides. For example, the antisense strand comprises deoxyribonucleotides at at least five positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand comprises deoxyribonucleotides at at least five positions selected from positions 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some other embodiments, the antisense strand contains at least six deoxyribonucleotides. For example, the antisense strand contains deoxyribonucleotides at at least six positions selected from positions 2, 5, 6, 7, 8, 9, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. Preferably, the antisense strand contains deoxyribonucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5'-end of the antisense strand hybridizing portion. In some other embodiments, deoxyribonucleotides are contained at one to four (preferably two, three, or four) positions selected from positions 2, 5, 7, and 12. In some other embodiments, deoxyribonucleotides are contained at one or two positions selected from positions 5 and 7. In some other embodiments, the antisense strand may not contain deoxyribonucleotides.

[0204] The sense strand of a dsRNA may contain at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) deoxyribonucleotide. In some embodiments, the sense strand contains 1, 2, 3, 4, or 5 deoxyribonucleotides. Without limitation, deoxyribonucleotides in the sense strand may be located at any position. In some embodiments, the sense strand contains at least one deoxyribonucleotide. For example, the sense strand contains a deoxyribonucleotide at one position selected from 11, 12, 13, or 15, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand contains a deoxyribonucleotide at position 11, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand contains a deoxyribonucleotide at position 12, counting from the 3'-end of the sense strand hybridizing portion. In some embodiments, the sense strand comprises a deoxyribonucleotide at position 13, counting from the 3'-end of the sense strand-hybridizing portion. In some embodiments, the sense strand comprises at least two deoxyribonucleotides. For example, the sense strand comprises deoxyribonucleotides at two positions selected from positions 11, 12, 13, or 15, counting from the 3'-end of the sense strand-hybridizing portion. In some embodiments, the antisense strand comprises at least three deoxyribonucleotides. For example, the sense strand comprises deoxyribonucleotides at three positions selected from positions 11, 12, 13, or 15, counting from the 3'-end of the sense strand-hybridizing portion. In some other embodiments, the sense strand comprises at least four deoxyribonucleotides. For example, the sense strand comprises deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3'-end of the sense strand-hybridizing portion. In another aspect, the sense strand may not comprise deoxyribonucleotides.

[0205] In some embodiments, the sense strand comprises a deoxyribonucleotide at at least one position opposite or complementary to position 11, 12, or 13, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the sense strand comprises a deoxyribonucleotide at at least one position opposite or complementary to position 11, 12, or 15, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises a deoxyribonucleotide at at least one position opposite or complementary to position 11, 12, 13, or 15, counting from the 5' end of the antisense strand hybridizing portion.

[0206] In some embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 12, and 13, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2 and 14, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In yet some other embodiments, the sense strand comprises deoxyribonucleotides at least at positions 11, 13, and 15 from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end of the antisense strand hybridizing portion.

[0207] In some embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion. In yet some other embodiments, the sense strand comprises deoxyribonucleotides at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand hybridizing portion, and the antisense strand comprises deoxyribonucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand hybridizing portion.

[0208] In some embodiments, the antisense strand does not contain deoxyribonucleotides at positions 3-9 counting from the 5' end of the antisense strand hybridizing portion.

[0209] In some embodiments, the dsRNA comprises one or more overhang regions (i.e., single-stranded regions) and / or capping groups at the 3'-end or 5'-end or both ends of the strand. Without limitation, the overhangs can be 1-10 nucleotides, 1-6 nucleotides, 1-5 nucleotides, 1-4 nucleotides, 1-3 nucleotides, 2-6 nucleotides, 2-5 nucleotides, 2-4 nucleotides, 2-3 nucleotides, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other or the result of two strands of the same length being offset. The overhangs can form mismatches with the target sequence, or they can be complementary to the target sequence or other sequences. The sense and antisense strands can be linked, for example, by forming a hairpin with additional nucleotides or by other non-nucleotide linkers. Without limitation, the overhangs can be present at the 3'-end of the sense strand, the antisense strand, or both strands.

[0210] In some embodiments, the dsRNA comprises a single overhang. For example, the dsRNA has a single overhang, and the overhang is at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the overhang is present at the 3'-end of the antisense strand. In certain embodiments, the dsRNA comprises a 2-nucleotide overhang at the 3'-end of the antisense strand. In certain embodiments, the dsRNA comprises a 3-nucleotide overhang at the 3'-end of the antisense strand. In certain embodiments, the dsRNA comprises a 4-nucleotide overhang at the 3'-end of the antisense strand.

[0211] dsRNA can also have blunt ends. For example, one end of the dsRNA is blunt and the other end has an overhang. Without limitation, the blunt end can be located at the 5' end of the antisense strand (or the 3' end of the sense strand), or vice versa. Generally, the antisense strand of a dsRNA has a nucleotide overhang at the 3' end and a blunt 5' end. For example, a dsRNA with an asymmetric blunt end at the 5' end of the antisense strand and an overhang at the 3' end of the antisense strand favors the process of loading the guide strand into RISC. In some embodiments, the dsRNA has a 2-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA has a 3-nucleotide overhang at the 3' end of the antisense strand and a blunt end at the 5' end of the antisense strand. In some embodiments, the dsRNA has a 4-nucleotide overhang on the 3'-end of the antisense strand and a blunt end on the 5'-end of the antisense strand.

[0212] In some other embodiments, the dsRNA has two blunt ends, ie, on both ends of the dsRNA.

[0213] The nucleotides in the overhang region of a dsRNA can each independently be modified or unmodified nucleotides, including, but not limited to, sugar-modified nucleotides, deoxyribonucleotides, ribonucleotides, and any combination thereof. In some other embodiments, nucleotides include 2'-O-XCE nucleotides, 2'-fluoro nucleotides, 2'-O-methyl nucleotides, deoxyribonucleotides, ribonucleotides, 2'-O-methoxyethyl nucleotides, and any combination thereof. For example, the base sequence TT (or UU) can be an overhang sequence for either end of either strand. The 5'- or 3'-overhang of the sense strand, the antisense strand, or both strands of a dsRNA can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate bond between them, where the two nucleotides in the overhang region can be the same or different.

[0214] dsRNA may contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more modified phosphodiester bonds. In some other embodiments, it may contain phosphorothioate bonds. Modified phosphodiester bonds may occur at any nucleotide at any position of the sense strand or antisense strand, or both strands. For example, modified phosphodiester bonds may occur at every nucleotide on the sense strand and / or antisense strand, or each modified phosphodiester bond may occur in an alternating pattern on the sense strand or antisense strand, or the sense strand or antisense strand contains both modified phosphodiester bonds in an alternating pattern. The alternating pattern of modified phosphodiester bonds on the sense strand may be the same or different from that of the antisense strand, and the alternating pattern of modified phosphodiester bonds on the sense strand may have a change to the alternating pattern of modified phosphodiester bonds on the antisense strand.

[0215] In some embodiments, dsRNA may or may not comprise modified phosphodiester bond in overhang region, but preferably comprises modified phosphodiester bond.In some other embodiments, dsRNA may comprise phosphorothioate bond in overhang region.For example, overhang region comprises two nucleotides with modified phosphodiester bond between them.Modified phosphodiester bond can also be provided to connect overhang nucleotide with paired nucleotide at the end of hybridization part.For example, at least 2, 3, 4 or all overhang nucleotides can be linked through modified phosphodiester bond, and optionally there can be additional modified phosphodiester bond that connects overhang nucleotide with paired nucleotide adjacent to overhang nucleotide.For example, there can be at least two modified phosphodiester bond between the three nucleotides at the end, where two nucleotides of the three are overhang nucleotide, and the third is paired nucleotide adjacent to overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3' end of the antisense strand.

[0216] In some embodiments, the dsRNA comprises one or more modified phosphodiester linkages within positions 1 to 10 of the termini of the sense and / or antisense strand, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides comprise modified phosphodiester linkages at one or both ends of the sense and / or antisense strand.

[0217] In some embodiments, the dsRNA comprises 1 to 5 modified phosphodiester linkages within positions 1 to 5 from the 5' end of the sense strand, 1 to 5 modified phosphodiester linkages within positions 1 to 5 from the 3' end of the sense strand, and 1 to 5 modified phosphodiester linkages within positions 1 to 5 from the 5' end of the antisense strand and 1 to 5 modified phosphodiester linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0218] In some embodiments, the dsRNA comprises 1 to 5 phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, 1 to 5 modified phosphorothioate linkages within positions 1 to 5 from the 3' end of the sense strand, and 1 to 5 phosphorothioate linkages within positions 1 to 5 from the 5' end of the antisense strand and 1 to 5 phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0219] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, one modified phosphorothioate linkage within positions 1 to 5 from the 3' end of the sense strand, and one phosphorothioate linkage within positions 1 to 5 from the 5' end of the antisense strand and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0220] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, two modified phosphorothioate linkages within positions 1 to 5 from the 3' end of the sense strand, and one phosphorothioate linkage within positions 1 to 5 from the 5' end of the antisense strand and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0221] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, two modified phosphorothioate linkages within positions 1 to 5 from the 3' end of the sense strand, and one phosphorothioate linkage within positions 1 to 5 from the 5' end of the antisense strand and one phosphorothioate linkage within positions 1 to 5 from the 3' end of the antisense strand.

[0222] In some embodiments, the dsRNA comprises one phosphorothioate linkage within positions 1 to 5 from the 5' end of the sense strand, one modified phosphorothioate linkage within positions 1 to 5 from the 3' end of the sense strand, and two phosphorothioate linkages within positions 1 to 5 from the 5' end of the antisense strand and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0223] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, two modified phosphorothioate linkages within positions 1 to 5 from the 3' end of the sense strand, and two phosphorothioate linkages within positions 1 to 5 from the 5' end of the antisense strand and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0224] In some embodiments, the dsRNA contains one phosphorothioate linkage within positions 1 to 5 from the 5' end of the sense strand, two at positions 1 and 2 from the 5' end of the antisense strand, and one phosphorothioate linkage within positions 1 to 5 from the 3' end of the antisense strand.

[0225] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, two phosphorothioate linkages at positions 1 and 2 from the 5' end of the antisense strand, and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0226] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, one phosphorothioate linkage within positions 1 to 5 from the 3' end of the sense strand, two phosphorothioate linkages at positions 1 and 2 from the 5' end of the antisense strand, and one phosphorothioate linkage within positions 1 to 5 from the 3' end of the antisense strand.

[0227] In some embodiments, the dsRNA comprises two phosphorothioate linkages within positions 1 to 5 from the 5' end of the sense strand, one phosphorothioate linkage within positions 1 to 5 from the 3' end of the sense strand, two phosphorothioate linkages at positions 1 and 2 from the 5' end of the antisense strand, and two phosphorothioate linkages within positions 1 to 5 from the 3' end of the antisense strand.

[0228] In some embodiments, the dsRNA comprises two phosphorothioate linkages at positions 1 and 2 counting from the 5' end of the sense strand, two phosphorothioate linkages at positions 1 and 2 counting from the 3' end of the sense strand, one phosphorothioate linkage at position 1 counting from the 5' end of the antisense strand, and one phosphorothioate linkage at position 1 counting from the 3' end of the antisense strand.

[0229] In some embodiments, the dsRNA comprises one phosphorothioate linkage at position 1 counting from the 5' end of the sense strand, one phosphorothioate linkage at position 1 counting from the 3' end of the sense strand, two phosphorothioate linkages at positions 1 and 2 counting from the 5' end of the antisense strand, and two phosphorothioate linkages at positions 1 and 2 counting from the 3' end of the antisense strand.

[0230] In some exemplary dsRNAs, the sense strand can contain 0, 1, 2, 3, or 4 phosphorothioate linkages. For example, the sense strand contains phosphorothioate linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 5' end of the sense strand.

[0231] In some exemplary dsRNA, antisense strand can comprise 1, 2, 3 or 4 phosphorothioate bonds.For example, sense strand comprises phosphorothioate bond between nucleotide position 1 and 2 and between nucleotide position 2 and 3, counting from the 3' end of sense strand.In another example, antisense strand comprises phosphorothioate bond between nucleotide position 1 and 2, between nucleotide position 2 and 3, counting from the 5' end of antisense strand, and between nucleotide position 1 and 2 and between nucleotide position 2 and 3, counting from the 3' end of antisense strand.

[0232] In some embodiments, the sense strand comprises phosphorothioate linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 5' end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 3' end of the antisense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 5' end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, counting from the 5' end of the antisense strand, and between nucleotide positions 1 and 2 and between nucleotide positions 2 and 3, counting from the 3' end of the antisense strand.

[0233] In some embodiments, the sense strand and / or antisense strand of the dsRNA may be 5' phosphorylated or may include a phosphate modification at the 5' end. Exemplary phosphate groups or modified phosphate groups include those compatible with RISC-mediated gene silencing. For example, suitable phosphate groups or modified phosphate groups include 5'-monophosphate ((HO) ２ (O)P-O-5'), 5'-diphosphate ((HO) ２ (O)P-O-P(HO)(O)-O-5'), 5'-triphosphate ((HO) ２ (O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate; (HO) ２(S)P—O-5′), 5′-monodithiophosphate (dithiophosphate, (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO) ２ (O)P-S-5'), any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-α-thiotriphosphate, 5'-γ-thiotriphosphate, etc.), 5'-phosphoramidic acid ((HO) ２ (O)P-NH-5', (HO)(NH ２ ) (O)P—O-5′), 5′-alkyl phosphonates (alkylene phosphates (e.g., (HO) ２ (O) P-CH ２ -5' (methylene phosphate), (HO) ２ (O) P-CH ２ CH ２ -5' (ethylene phosphate), R'P(OH)(O)-O-5'- (R' = alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-cycloalkylphosphonate ((HO) ２ (O) P-C ３ H ４ -5'(cyclopropylene phosphate)), 5'-alkenyl phosphonate (alkenyl is, for example, vinyl ((OH) ２ Modifications include 5'-alkoxyalkyl phosphonates (R"P(OH)(O)-O-5'- (R" = alkoxyalkyl, e.g., methoxymethyl, ethoxymethyl, etc.)). Modifications may be placed within the antisense strand of the dsRNA. For example, the antisense strand may contain a 5' phosphonate group at the 5' end, or may contain 5'-vinyl phosphonate nucleotides, 5'-ethyl phosphonate nucleotides, or 5'-cyclopropane phosphonate nucleotides.

[0234] In some embodiments, the 5'-terminal nucleotide of the antisense strand comprises a 5'-E-vinylphosphonate, for example, the following formula (II-3): (Base in formula (II-3) has the same meaning as X and Base in formula (II)) (5'-vinylphosphonate (VP)-modified 2'-O-Me nucleotide) is preferred as the 5'-E-vinylphosphonate.

[0235] In some other embodiments, the following formula (II-2): (X and Base in formula (II-2) have the same meanings as X and Base in formula (II)) (5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide) is preferred.

[0236] In some embodiments, the following formula (III-3): (Base in formula (III-3) has the same meaning as X and Base in formula (III)) (5'-cyclopropanephosphonate (CPP)-modified 2'-O-Me nucleotide) is preferred.

[0237] In some other embodiments, the compound of formula (III-2) below: (X and Base in formula (III-2) have the same meanings as X and Base in formula (III)) (5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide) is preferred.

[0238] In some other embodiments, the compound of formula (IV-3) below: (Base in formula (IV-3) has the same meaning as X and Base in formula (IV)) (5'-ethylphosphonate (EP)-modified 2'-O-Me nucleotide) is preferred.

[0239] In some other embodiments, the compound of formula (IV-2) below: (X and Base in formula (IV-2) have the same meanings as X and Base in formula (IV)) A structure (5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide) is preferred. The wavy lines in formulas (II-2) to (IV-3) are understood to represent the bonding position of the internucleotide bond with the adjacent nucleotide, the bonding position with a functional molecule or its linker, the bonding position with a hydrogen atom, a hydroxy-protecting group or a phosphorus-containing group, etc.

[0240] In some embodiments, the sense strand comprises a 5'-morpholino modification, a 5'-dimethylamino modification, a 5'-deoxy modification, an inverted abasic modification, or an inverted abasic locked nucleic acid modification at the 5' end.

[0241] Exemplary nucleoside structures used as 5'-morpholino modifications include, but are not limited to, those of formula (XVI) below: (Base in formula (XVI) has the same meaning as Base in formula (I), and R ４０ is a hydrogen atom, a hydroxy group, a C1-6 alkoxy group, fluorine, an amino group, an N-methylamino group, an N,N'-dimethylamino group, a 2-methoxyethoxy group (O-MOE), a 2-(N-methylcarbamoyl)ethoxy group (O-MCE), DMAECE, MorECE, PyECE, and BimECE).

[0242] Exemplary nucleoside structures used as 5'-dimethylamino modifications include, but are not limited to, the following formula (XVII): (Base in formula (XVII) has the same meaning as Base in formula (I), and R ４０ is R in formula (XVI) ４０ (synonymous with "anti-

[0243] Exemplary nucleoside structures used as 5'-deoxy modifications include, but are not limited to, the following formula (XVIII): (Base in formula (XVIII) has the same meaning as Base in formula (I), and R ４０ is R in formula (XVI) ４０ (synonymous with "anti-

[0244] Exemplary nucleoside structures used as inverted abasic modifications include, but are not limited to, those of the following formula (XIX): (wherein the wavy line indicates the binding position to the 5' position of the adjacent nucleotide).

[0245] Exemplary nucleoside structures for use as inverted abasic locked nucleic acid modifications include, but are not limited to, those of the following formula (XX): (wherein the wavy line indicates the binding position to the 5' position of the adjacent nucleotide).

[0246] Generally, dsRNAs have a melting temperature (Tm) in the range of about 40°C to about 80°C. For example, dsRNAs have a melting temperature at the low end of the range from about 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C and at the high end of the range from about 70°C, 75°C, or 80°C. In some embodiments, dsRNAs have a melting temperature in the range of about 55°C to about 70°C or in the range of about 60°C to about 75°C. In some embodiments, dsRNAs have a melting temperature in the range of about 57°C to about 67°C. In some specific embodiments, dsRNAs have a melting temperature in the range of about 60°C to about 67°C. In some further embodiments, dsRNAs have a melting temperature in the range of about 62°C to about 66°C.

[0247] For example, dsRNAs that have a melting temperature of at least 60°C are more effective in vivo and in vitro. Thus, in some embodiments, the dsRNA has a melting temperature of at least 60°C.

[0248] For example, a thermodestabilizing modification at positions 2-9, counting from the 5' end of the antisense strand hybridizing portion, can reduce or inhibit off-target gene silencing. Thus, in some embodiments, the antisense strand comprises at least one (e.g., 1, 2, 3, 4, 5, or more) thermodestabilizing modification of the duplex within 9 nucleotide positions, counting from the 5' end of the antisense strand hybridizing portion. The term "one or more thermodestabilizing modifications" includes one or more modifications that will result in a dsRNA with an overall lower melting temperature (Tm) (preferably a Tm that is 1, 2, 3, or 4 degrees lower) than the Tm of a dsRNA lacking such one or more modifications.

[0249] In some embodiments, the thermodestabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8, or 9, or preferably position 4, 5, 6, 7, or 8, counting from the 5' end of the antisense strand hybridizing portion. In some embodiments, the thermodestabilizing modification is located at position 2, 3, 4, 5, or 9, counting from the 5' end of the antisense strand hybridizing portion. In some other embodiments, the thermodestabilizing modification is located at position 6, 7, or 8, counting from the 5' end of the antisense strand hybridizing portion. In some particular embodiments, the thermodestabilizing modification is located at position 7, counting from the 5' end of the antisense strand hybridizing portion.

[0250] In some embodiments, the antisense strand comprises at least one 2'-modified nucleotide or 2'-4'-bridged nucleotide adjacent to the thermally destabilizing modification. For example, the 2'-modified nucleotide or 2'-4'-bridged nucleotide can be at the 5'-end or 3'-end of the thermally destabilizing modification, i.e., the nucleotide at position -1 or +1 from the position of the thermally destabilizing modification. In some embodiments, the antisense strand comprises a 2'-modified nucleotide or 2'-4'-bridged nucleotide at each of the 5'-end and 3'-end of the thermally destabilizing modification, i.e., the -1 and +1 positions from the position of the destabilizing modification.

[0251] In some embodiments, the antisense strand comprises at least two modified nucleotides independently selected from a 2'-modified nucleotide or a 2'-4' bridged nucleotide at the 3' end of the thermally destabilizing modification, i.e., at positions +1 and +2 from the position of the thermally destabilizing modification.

[0252] In some embodiments, the sense strand does not contain a 2'-modified nucleotide or a 2'-4'-bridged nucleotide at the position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0253] In some embodiments, the antisense strand comprises at least one 2'-fluoro nucleotide adjacent to the thermally destabilizing modification. For example, the 2'-fluoro nucleotide can be at the 5' or 3' end of the thermally destabilizing modification, i.e., the nucleotide at position -1 or +1 from the position of the thermally destabilizing modification. In some embodiments, the antisense strand comprises a 2'-fluoro nucleotide at each of the 5' and 3' ends of the thermally destabilizing modification, i.e., the -1 and +1 positions from the position of the destabilizing modification.

[0254] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the thermally destabilizing modification, ie, at positions +1 and +2 from the position of the thermally destabilizing modification.

[0255] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at the position opposite or complementary to a thermally destabilizing modification of the duplex in the antisense strand.

[0256] In some embodiments, it may be possible to include particular bases in the overhang or to include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' or 3' overhang, or both. For example, it may be desirable to include purine nucleotides in the overhang. In some embodiments, all or a portion of the bases in the 3' or 5' overhang may be modified, e.g., with the modifications described herein. Modifications may include, for example, the use of 2'-modified nucleotides, e.g., 2'-O-XCE nucleotides, 2'-fluoro nucleotides, or 2'-O-Me nucleotides, and modified phosphate groups, e.g., phosphorothioate linkages. The overhang need not be homologous to the target sequence.

[0257] In some embodiments, each residue in the sense strand and the antisense strand is independently modified with a 2' modification (such as 2'-O-MOE, 2'-O-Me, or 2'-fluoro), a 2'-4' bridge modification (such as LNA), or a 2'-deoxy. Each strand may contain more than one modification. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with 2'-O-Me or 2'-fluoro. It should be understood that these modifications may be present in a dsRNA in addition to one or more 2'-O-XCE nucleotides.

[0258] In some embodiments, in addition to one or more 2'-O-XCE nucleotides, there may be at least one (preferably two) different modified nucleotides on the sense strand and / or antisense strand. Each of these modifications may be a deoxyribonucleotide, a 2'-O-Me nucleotide, a 2'-fluoro nucleotide, an acyclic nucleotide, etc. In some embodiments, the sense strand and the antisense strand each comprise one or two different modified nucleotides selected from 2'-O-Me nucleotides, 2'-fluoro nucleotides, and / or 2'-deoxyribonucleotides. In some embodiments, the sense and antisense strands are independently modified with 2'-O-Me nucleotides, deoxyribonucleotides, 2'-fluoro nucleotides, 2'-O-N-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides (also referred to as 2'-fluoroarabinonucleotides, 2'-F-ANA). Again, it should be understood that these modifications are present in addition to one or more 2'-O-XCE nucleotides in a dsRNA.

[0259] In some embodiments, dsRNA can comprise alternating pattern modifications.The term "alternating pattern" as used herein refers to a modification pattern in which one strand has one or more modifications, and each modified nucleotide has a repeat unit that alternates, and the number of repeats is two or more.The nucleotide that appears in alternating pattern can appear every other or every third nucleotide.For example, when A, B and C each represent one type of modification for nucleotide, the alternating pattern with two repeats includes "ABAB", "AABBAABB", "AABAAB", "AAABAAAB", "AAABBBAAABBB" or "ABCABC" etc. In each strand the alternating pattern can be "ABABABABABAB...", "AABBAABBAABB...", "AABAABAABAAB...", "AAABAAABAAAB...", "AAABBBAAABBB..." or "ABCABCABCABC...", etc.

[0260] The types of modifications included in the alternating pattern can be the same or different. For example, if A, B, C, and D each represent one type of modification on a nucleotide, the alternating pattern, i.e., the modification of every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possibilities of modifications within the alternating pattern, such as "ABAB...", "ACAC...", "BDBD..." or "CDCD...".

[0261] In some embodiments, the dsRNA comprises an alternation pattern on the sense strand that is shifted relative to the alternation pattern on the antisense strand. This shift can be such that a group of modified nucleotides on the sense strand corresponds to a differently modified group of nucleotides on the antisense strand, and vice versa. For example, when the sense strand pairs with the antisense strand in a dsRNA duplex, the alternation pattern on the sense strand can begin with "ABABAB" at the 5'-3' end of the strand, and the alternation pattern on the antisense strand can begin with "BABABA" at the 3'-5' end of the strand, within the hybridizing portion. As another example, the alternation pattern on the sense strand can begin with "AABBAABB" at the 5'-3' end of the strand, and the alternation pattern on the antisense strand can begin with "BBAABBAA" at the 3'-5' end of the strand, within the hybridizing portion, thus resulting in a complete or partial shift in modification patterns between the sense and antisense strands.

[0262] In some embodiments, dsRNA contains one or more mismatches with target RNA within the double strand or combinations thereof. Mismatches can appear in overhanging regions or hybridized portions. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., regarding the free energy of association or dissociation of a particular pairing, the simplest method is to examine the pairs on each base pair, but adjacent base analysis or similar analysis can also be used). In terms of promoting dissociation, A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, such as non-standard or non-standard pairings (as described elsewhere herein), are preferred over standard (A:T, A:U, G:C) pairings; and pairings involving universal bases are preferred over standard pairings.

[0263] In some embodiments, the dsRNA contains at least one of the first 1, 2, 3, 4, or 5 base pairs (which may be independently selected from the group of A:U, G:U, I:C) counting from the 5' end of the antisense strand hybridizing portion, and a mismatch pair, e.g., a non-standard or non-standard pairing or a pairing containing a universal base, to promote dissociation of the antisense strand at the 5' end of the duplex.

[0264] In some embodiments, the nucleotide at position 1, counting from the 5' end of the antisense strand hybridizing portion, is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pairs, counting from the 5' end of the antisense strand hybridizing portion, is an AU base pair. For example, the first base pair, counting from the 5' end of the antisense strand hybridizing portion, is an AU base pair.

[0265] For example, the introduction of 4'- and / or 5'-modified nucleotides at the 3' end of a phosphodiester, phosphorothioate and / or phosphorodithioate linkage of a dinucleotide at any position in a single- or double-stranded oligonucleotide can exert a steric effect on the internucleotide bond, thus protecting or stabilizing it from nucleases.

[0266] In some embodiments, a 5'-modified nucleotide is introduced at the 3' end of a dinucleotide at any position in a dsRNA. For example, a 5'-alkylated nucleotide can be introduced at the 3' end of a dinucleotide at any position in a dsRNA.

[0267] In some embodiments, a 4'-modified nucleotide is introduced at the 3'-end of a dinucleotide at any position in a dsRNA. For example, a 4'-alkylated nucleotide may be introduced at the 3'-end of a dinucleotide at any position in a dsRNA. Alternatively, a 4'-O-alkylated nucleotide may be introduced at the 3'-end of a dinucleotide at any position in a single-stranded or double-stranded siRNA. An exemplary 4'-O-alkylated nucleotide is a 4'-O-methyl nucleotide. The 4'-O-methyl may be either racemic or chirally pure R or S isomer.

[0268] In some embodiments, 5'-alkylated nucleotides are introduced at any position on the sense or antisense strand of a dsRNA, and such modifications maintain or improve the potency of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomers. An exemplary 5'-alkylated nucleotide is a 5'-methyl nucleotide. The 5'-methyl can be either racemic or chirally pure R or S isomers. Another exemplary 5'-alkylated nucleotide is a 5'-CP nucleotide.

[0269] In some embodiments, 4'-alkylated nucleotides are introduced at any position on the sense or antisense strand of a dsRNA, where such modifications maintain or improve the potency of the dsRNA. The 4'-alkyl can be either racemic or chirally pure R or S isomers. An exemplary 4'-alkylated nucleotide is a 4'-methyl nucleotide. The 4'-methyl can be either racemic or chirally pure R or S isomers.

[0270] In some embodiments, 4'-O-alkylated nucleotides are introduced at any position on the sense or antisense strand of a dsRNA, where such modifications maintain or improve the potency of the dsRNA. The 5'-alkyl can be either racemic or chirally pure R or S isomers. An exemplary 4'-O-alkylated nucleotide is a 4'-O-methyl nucleotide. The 4'-O-methyl can be either racemic or chirally pure R or S isomers.

[0271] In some embodiments, dsRNAs can contain 2'-5' linkages (including 2'-H, 2'-OH, and 2'-O-Me and including P=O or P=S). For example, 2'-5' linkage modifications can be used to promote nuclease resistance, or to inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent activation of the sense strand by RISC. In some embodiments, the sense strand contains a 2'-5' linkage between the 1st and 2nd nucleotides counting from the 5' end.

[0272] In some embodiments, functional molecule can be directly or indirectly bound to dsRNA.The binding between functional molecule and sense strand or / and antisense strand of dsRNA can be direct or indirect through other substances, but preferably oligonucleotide and functional molecule are bound by covalent bond, ionic bond or hydrogen bond.From the viewpoint of high stability of this binding, it is more preferred that they are bound by covalent bond directly or by covalent bond via linker (linking group).

[0273] When the functional molecule is covalently bound to the sense strand or / and antisense strand of the dsRNA, the functional molecule is preferably bound directly or indirectly to the 3'-end or 5'-end of the sense strand or / and antisense strand of the dsRNA. In some embodiments, the functional molecule is preferably bound directly or indirectly to an internal nucleotide (e.g., sugar moiety, base moiety, or internucleotide linkage) of the sense strand or / and antisense strand of the dsRNA. The bond between the linker or functional molecule and the terminal nucleotide or internal nucleotide of the sense strand or / and antisense strand of the dsRNA is selected depending on the functional molecule. The linker or functional molecule is preferably linked to the terminal nucleotide or internal nucleotide of the sense strand or / and antisense strand of the dsRNA via a phosphodiester bond or a modified phosphodiester bond, more preferably via a phosphodiester bond. In some embodiments, the linker or functional molecule is preferably linked to the terminal nucleotide of the sense strand or / and antisense strand of the dsRNA via a modified phosphodiester bond, more preferably via a phosphorothioate bond. The linker or functional molecule may be directly linked to the 3' oxygen atom of the 3'-terminal nucleotide or the 5' oxygen atom of the 5'-terminal nucleotide of the sense strand or / and antisense strand of the dsRNA. In some embodiments, the linker or functional molecule may be directly linked to the 2' oxygen atom of the terminal nucleotide or an internal nucleotide of the sense strand or / and antisense strand of the dsRNA.

[0274] The structure of the "functional molecule" is not particularly limited, and the binding thereof confers a desired function to the dsRNA. Desired functions include labeling, purification, and delivery to a target site. Examples of molecules that impart a labeling function include compounds such as fluorescent proteins and luciferase. Examples of molecules that impart a purification function include compounds such as biotin, avidin, His tag peptide, GST tag peptide, and FLAG tag peptide.

[0275] In addition, from the viewpoint of highly specific and efficient delivery of dsRNA to target site (for example, target cell), and the expression of target RNA is controlled very effectively by this dsRNA, it is preferable that the molecule that has the function of delivering dsRNA to target site is bound as functional molecule.For the molecule that has this delivery function, for example, can refer to European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp 791-822 etc.

[0276] Molecules that confer delivery function to target RNA include, for example, lipids and sugars, which can deliver dsRNA with high specificity and efficiency to the liver, central tissue, lungs, etc. Examples of such lipids include cholesterol; fatty acids; fat-soluble vitamins such as vitamin E (tocopherols, tocotrienols), vitamin A, vitamin D, and vitamin K; intermediate metabolites such as acylcarnitine and acyl-CoA; glycolipids; glycerides; long-chain hydrocarbons; and derivatives thereof. Among these, cholesterol and vitamin E (tocopherols, tocotrienols) are preferred from the viewpoint of higher safety. Among these, tocopherols are more preferred, tocopherol is even more preferred, and α-tocopherol is particularly preferred. Examples of sugars include sugar derivatives that interact with the asialoglycoprotein receptor.

[0277] In some other embodiments, the following formula (XXI): (Base in formula (XXI) has the same meaning as Base in formula (I), and R ５０ is a C5-30 alkyl group or a C5-30 alkenyl group) is preferably contained in the sense strand or / and antisense strand of the dsRNA, and more preferably in the sense strand. In some other embodiments, it is preferable to contain formula (XXI) in at least one position selected from 14 to 21 counting from the 3' end of the sense strand hybridizing portion, and more preferably to contain formula (XXI) in 16 position. In some other embodiments, R ５０ is preferably a C14-24 alkyl group, and R ５０ More preferably, is a C16-22 alkyl group.

[0278] The "asialoglycoprotein receptor" is present on the surface of liver cells and recognizes the galactose residue of asialoglycoprotein, taking up the molecule into the cell and degrading it. The "sugar derivative that interacts with the asialoglycoprotein receptor" is preferably a compound that has a structure similar to that of a galactose residue and is taken up into the cell through interaction with the asialoglycoprotein receptor, and examples thereof include GalNAc (N-acetylgalactosamine) derivatives, galactose derivatives, and lactose derivatives.

[0279] In some other embodiments, the "saccharide derivative that interacts with the asialoglycoprotein receptor" is a sugar derivative of the following formula (i): (wherein Rd is a hydroxyl group or a thiol group, preferably a thiol group) is preferably contained in the sense strand or / and antisense strand of the dsRNA, more preferably in the sense strand. In some other embodiments, it is preferably directly bound to the 3'-end or 5'-end of the sense strand of the dsRNA, more preferably directly bound to the 5'-end of the sense strand.

[0280] In some other embodiments, the "asialoglycoprotein receptor-interacting sugar derivative" is a sugar derivative represented by the following formula (ii): (wherein each Re is independently a hydroxyl group or a thiol group, preferably a thiol group) is preferably contained in the sense strand or / and antisense strand of the dsRNA, more preferably in the sense strand. In some other embodiments, it is preferably directly bound to the 3'-end or 5'-end of the sense strand of the dsRNA, more preferably directly bound to the 3'-end of the sense strand.

[0281] In addition, from the viewpoint of being able to deliver the dsRNA of the present invention to the brain with high specificity and efficiency, the "functional molecule" can be exemplified by sugar (for example, glucose, sucrose, etc.).In addition, from the viewpoint of being able to deliver the dsRNA to the organ with high specificity and efficiency by interacting with various proteins on the cell surface of each organ, the "functional molecule" can be exemplified by receptor ligand, antibody, peptide or protein of their fragments.

[0282] The linker that connects the functional molecule to the sense strand and / or antisense strand of the dsRNA is not particularly limited as long as it can exhibit the function of the functional molecule as a dsRNA and can stably link the functional molecule to the oligonucleotide. Examples of such linkers include groups derived from oligonucleotides containing 1 to 20 nucleotides, groups derived from polypeptides containing 2 to 20 amino acids, alkylenes containing 2 to 20 carbon atoms, and alkenylenes containing 2 to 20 carbon atoms. The group derived from an oligonucleotide containing 2 to 20 nucleotides is a group obtained by removing a hydroxyl, hydrogen atom, or the like from an oligonucleotide containing 2 to 20 nucleotides. For examples of groups derived from oligonucleotides containing 1 to 20 nucleotides, see International Publication No. 2017 / 053995. International Publication No. 2017 / 053995 describes, for example, a 3-base linker having a TCA motif and a 1-5-base linker without a TCA motif. The group derived from a polypeptide having 2 to 20 amino acids is a group obtained by removing a hydroxyl, hydrogen atom, amino, or the like from a polypeptide having 2 to 20 amino acids.

[0283] The linker is preferably a C2-20 alkylene or a C2-20 alkenylene (methylenes contained in the alkylene and alkenylene are each independently unsubstituted or substituted with one or two substituents selected from the group consisting of a halogen atom, hydroxy, protected hydroxy, oxo, and thioxo. In addition, methylenes in the alkylene and alkenylene are each independently unsubstituted or substituted with —O—, —NR Ｂ - (R Ｂ is a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -S-, -S(=O)- or -S(=O) ２ In this case, by combining the above substitutions, the linker is -C(=O)-O-, -O-C(=O)-NR ２３ - (R ２３ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), —C(═O)—NR ２３ - (R ２３ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), -C(=S)-NR ２３ - (R ２３ represents a hydrogen atom, C1-6 alkyl or haloC1-6 alkyl), —NR ２３ —C(═O)—NR ２３ - (R ２３ each independently represents a hydrogen atom, a C1-6 alkyl, or a haloC1-6 alkyl). For example, —[C2-6 alkylene]-[C(═O)—NR ２３ - (R ２３ represents a hydrogen atom or a C1-6 alkyl)]-[C2-6 alkylene]-[C(=O)]- can be used. Here, preferably, the left side [C2-6 alkylene] binds to the functional molecule, and the right side [C(=O)] binds to the oligonucleotide.

[0284] The linker is more preferably a C2-20 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by —O—; each unsubstituted methylene is independently unsubstituted or substituted by hydroxy or protected hydroxy), and even more preferably a C8-12 alkylene (wherein each methylene of the alkylene is independently unsubstituted or replaced by —O—; each unsubstituted methylene is independently unsubstituted or substituted by hydroxy).

[0285] In another embodiment, the linker is more preferably a C2-20 alkylene (each methylene of the alkylene is independently unsubstituted or -O- or -NR Ｂ - (R Ｂ is a hydrogen atom or a C1-6 alkyl). Each unsubstituted methylene is independently unsubstituted or substituted by oxo), and more preferably C8-12 alkylene (each methylene of the alkylene is independently unsubstituted or substituted by —O— or —NR Ｂ - (R Ｂ is replaced by a hydrogen atom or a C1-6 alkyl. Each unsubstituted methylene is independently unsubstituted or substituted by oxo.

[0286] One to five (preferably one to three, particularly preferably three) 3-11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol structures may be linked via phosphodiester or phosphorothioate bonds to the 5'-end or 3'-end of the sense strand and / or antisense strand of the dsRNA, and the linker may be linked to the 3-11-membered nitrogen-containing non-aromatic heterocycle. In this case, the number of functional molecules linked via the linker is also preferably one to five, more preferably one to three, and particularly preferably three. The 3-11-membered nitrogen-containing non-aromatic heterocycle is preferably pyrrolidine or piperidine. The 3-11-membered nitrogen-containing non-aromatic heterocycle-diyldimethanol is particularly preferably piperidine-4,4-diylmethanol.

[0287] The protecting group for the "protected hydroxy" is not particularly limited as long as it is stable when the functional molecule and the oligonucleotide are linked. The linker is not particularly limited, and examples thereof include any protecting group described in Protective Groups in Organic Synthesis, 4th Edition, by T.W. Greene and P.G.M. Wuts, John Wiley & Sons Inc. (2006). Specifically, as described in the explanation of terms, preferred are benzoyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, triphenylmethyl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, more preferably monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl, and even more preferably dimethoxytrityl. The linker can be linked to a mer.

[0288] In a dsRNA, the functional molecule can be attached to one or both strands. In some embodiments, the dsRNA has the functional molecule attached to the sense strand. In other embodiments, the dsRNA has the functional molecule attached to the antisense strand.

[0289] In some embodiments, functional molecules can be attached to the nucleobase, sugar moiety, or internucleoside linkage of the sense strand or / and antisense strand of a dsRNA. Attachment to a purine nucleobase or derivative thereof can occur at any position, including the endocyclic and exocyclic atoms. In some embodiments, attachment is to the 2-, 6-, 7-, or 8-position of a purine nucleobase. Attachment to a pyrimidine nucleobase or derivative thereof can also occur at any position. In some embodiments, attachment can be to the 2-, 5-, or 6-position of a pyrimidine nucleobase. Attachment to the sugar moiety of a nucleotide can occur at any carbon atom. Examples of carbon atoms of the sugar moiety that can be attached include the 2', 3', and 5' carbon atoms. The 1' position can also be attached, for example, at an abasic residue. Internucleoside linkages can also have conjugate moieties. Phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate, dithiophosphate, phosphoramidate, etc.) can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In amine or amide containing internucleoside linkages (eg, PNA), attachment can be to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0290] In some embodiments, the functional molecule is attached to the sense strand. As described herein, the functional molecule can be attached to the 3'-end, 5'-end, or an internal position of the sense strand. In some embodiments, the functional molecule is attached to the 3'-end of the sense strand. Furthermore, the functional molecule can be attached to a nucleobase, sugar moiety, or internucleotide linkage of the sense strand.

[0291] dsRNA not only exists through their tautomerism and geometric isomerism, but also includes the mixture or mixture of each isomer.In addition, when there is an asymmetric center, or when an asymmetric center is generated as a result of isomerization, it also includes the existence of each optical isomer and a mixture of any ratio.In addition, when a compound has two or more asymmetric centers, there also exist diastereomers of each optical isomer.The present invention also includes all of these forms in any ratio.In addition, optically active forms can be obtained by well-known methods for this purpose.

[0292] For example, when the dsRNA of the present invention contains a modified phosphodiester bond (e.g., a phosphorothioate bond) and the phosphorus atom is an asymmetric atom, both oligonucleotides in which the phosphorus atom is stereoregulated and oligonucleotides in which the phosphorus atom is not stereoregulated are included within the scope of the present invention.

[0293] The dsRNA or pharmaceutically acceptable salt thereof of the present invention can exist in any crystalline form or any hydrate depending on the manufacturing conditions, and these crystalline forms, hydrates, and mixtures thereof are also included within the scope of the present invention. They may also exist as solvates containing organic solvents such as acetone, ethanol, 1-propanol, and 2-propanol, and all of these forms are included within the scope of the present invention.

[0294] The dsRNA of the present invention can be converted into a pharmaceutically acceptable salt as needed, or can be released from the resulting salt. Pharmaceutically acceptable salts of dsRNA include, for example, salts with alkali metals (lithium, sodium, potassium, etc.), alkaline earth metals (magnesium, calcium, etc.), ammonium, organic bases (triethylamine, trimethylamine, etc.), amino acids (glycine, lysine, glutamic acid, etc.), inorganic acids (hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, etc.) or organic acids (acetic acid, citric acid, maleic acid, fumaric acid, tartaric acid, benzenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, etc.). In particular, the partial structure represented by -P(=O)(OH)- is converted into an anionic partial structure represented by -P(=O)(O-)-, and may form a salt with an alkali metal (lithium, sodium, potassium, etc.), alkaline earth metal (magnesium, calcium, etc.), or ammonium, etc. Furthermore, the partial structure represented by -P(=O)(SH)-, which forms a phosphorothioate bond, may be converted into an anionic partial structure represented by -P(=O)(S-)-, and similarly form a salt with an alkali metal, alkaline earth metal, ammonium, etc. The same applies to other modified phosphodiester bonds.

[0295] Those skilled in the art can prepare the dsRNA of the present invention by appropriately selecting a known method. For example, those skilled in the art can design the nucleotide sequence of the dsRNA based on the information of the nucleotide sequence of the target RNA, and synthesize it using a commercially available automatic nucleic acid synthesizer (Applied Biosystems, Beckman, Gene Design, etc.). It can also be synthesized by a reaction using an enzyme. Examples of the enzyme include, but are not limited to, polymerase, ligase, and restriction enzymes. That is, the method for producing dsRNA according to this embodiment can include a step of extending the nucleotide chain at the 3'-end or 5'-end of the sense strand and / or antisense strand.

[0296] Many methods for linking functional molecules to the sense strand and / or antisense strand of the dsRNA are well known in the art, and reference can be made to, for example, European Journal of Pharmaceutics and Biopharmaceutics, 2016, 107, pp. 321-340, Advanced Drug Delivery Reviews, 2016, 104, pp. 78-92, Expert Opinion on Drug Delivery, 2014, 11, pp. 791-822, etc. For example, after linking the functional molecule to a linker by a known method, it can be derivatized into an amidite form using an amiditizing reagent or into an H-phosphonate form using an H-phosphonate reagent, and then linked to an oligonucleotide.

[0297] The sense and antisense strands of dsRNA can be prepared by purifying the resulting oligonucleotides by reverse phase column chromatography, etc. dsRNA can be prepared by mixing equal amounts of the sense and antisense strands and annealing them.

[0298] The dsRNA of the present invention can effectively control the expression of target RNA.Therefore, the present invention can provide the composition that contains the dsRNA of the present invention as active ingredient, for example, for controlling the expression of target RNA by RNA interference effect.In particular, the dsRNA of the present invention can obtain high efficacy by administration of low concentration, and in some embodiments, can also provide the pharmaceutical composition for treating, preventing and improving the diseases that are associated with the increased expression of target RNA, such as metabolic disease, tumor, infectious disease.

[0299] The composition comprising the dsRNA of the present invention can be prepared by known pharmaceutical methods.For example, it can be used as capsules, tablets, pills, liquids, powders, granules, fine granules, film coating agents, pellets, lozenges, sublinguals, chewable agents, buccal agents, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, cataplasms, transdermal preparations, lotions, inhalants, aerosols, injections, suppositories etc., enteral (orally etc.) or parenteral.

[0300] In preparing these formulations, the pharmaceutical composition may be appropriately combined with a carrier that is pharmacologically or acceptable as a food or beverage, specifically, sterilized water, physiological saline, vegetable oil, solvent, base, emulsifier, suspending agent, surfactant, pH adjuster, stabilizer, flavoring agent, fragrance, excipient, vehicle, preservative, binder, diluent, isotonic agent, soothing agent, bulking agent, disintegrant, buffer, coating agent, lubricant, colorant, sweetener, thickener, flavoring agent, solubilizing agent, or other additives.

[0301] The administration form of the composition that comprises the dsRNA of the present invention is not particularly limited, but can be enteral (oral etc.) or parenteral administration.More preferably, can be intravenous administration, intraarterial administration, intraperitoneal administration, subcutaneous administration, intradermal administration, intratracheal administration, rectal administration, intramuscular administration, intrathecal administration, intraventricular administration, intranasal administration and intravitreal administration etc. and administration by infusion.

[0302] Diseases that can be treated, prevented, or ameliorated by nucleic acid drugs using the dsRNA of the present invention are not particularly limited, and examples include diseases caused by gene expression, such as metabolic diseases, cardiovascular diseases, tumors, infectious diseases, eye diseases, inflammatory diseases, autoimmune diseases, and rare genetic diseases. More specifically, hypercholesterolemia, hypertriglyceridemia, spinal muscular atrophy, muscular dystrophy (Duchenne muscular dystrophy, myotonic dystrophy, congenital muscular dystrophy (Fukuyama congenital muscular dystrophy, Ullrich congenital muscular dystrophy, merosin-deficient congenital muscular dystrophy, integrin deficiency, Walker-Warburg syndrome, etc.), Becker muscular dystrophy, limb-girdle muscular dystrophy, Miyoshi muscular dystrophy, facioscapulohumeral muscular dystrophy, etc.), Huntington's disease, and rheumatoid arthritis. Examples of diseases that can be targeted include Ralzheimer's disease, transthyretin amyloidosis, familial amyloidotic cardiomyopathy, multiple sclerosis, Crohn's disease, inflammatory bowel disease, acromegaly, type 2 diabetes, chronic nephropathy, respiratory syncytial virus infection, Ebola hemorrhagic fever, Marburg fever, HIV, influenza, hepatitis B, hepatitis C, cirrhosis, chronic heart failure, myocardial fibrosis, atrial fibrillation, prostate cancer, melanoma, breast cancer, pancreatic cancer, colon cancer, renal cell carcinoma, bile duct cancer, cervical cancer, liver cancer, lung cancer, leukemia, non-Hodgkin's lymphoma, atopic dermatitis, glaucoma, and age-related macular degeneration. Depending on the type of disease, a gene that causes the disease is designated as the target RNA, and the expression control sequence (e.g., an antisense sequence) can be appropriately designated depending on the sequence of the target RNA.

[0303] In addition to primates such as humans, a variety of mammalian diseases can be treated, prevented, or ameliorated with compositions containing the double-stranded RNA of the present invention or its prodrug. For example, diseases can be treated in mammalian species, including, but not limited to, cows, sheep, goats, horses, dogs, cats, guinea pigs, and other bovine, ovine, equine, canine, feline, and rodent species such as mice. Compositions containing double-stranded RNA can also be used in other species, such as birds (e.g., chickens).

[0304] When a composition containing the dsRNA of the present invention is administered to or ingested by an animal, including a human, the dosage or intake is appropriately selected depending on the age, weight, symptoms, and health condition of the subject, the type of composition (e.g., a drug, a food or drink), etc., and the dosage or intake is preferably 0.0001 mg / kg / day to 100 mg / kg / day.

[0305] The dsRNA of the present invention can effectively control the expression of target RNA, and has higher metabolic stability than conventional siRNA.Therefore, it can provide the method of administering the dsRNA of the present invention to animals, including humans, and control the expression of target RNA for a longer period by RNA interference effect.In addition, it can also provide the method of treating, preventing and improving various diseases that involve the enhanced expression of target RNA, including by administering the composition that comprises the dsRNA of the present invention to animals, including humans.

[0306] Preferred methods for using the dsRNA of the present invention include the following: A method for regulating the function of a target RNA, comprising contacting a cell with the dsRNA of the present invention. A method for regulating the function of a target RNA in a mammal, comprising administering to the mammal a pharmaceutical composition comprising the dsRNA of the present invention. Use of the dsRNA of the present invention for regulating the function of a target RNA in a mammal. Use of the dsRNA of the present invention for producing a medicament for regulating the function of a target RNA in a mammal. A method for regulating the expression of a target RNA, comprising contacting a cell with the dsRNA of the present invention. A method for regulating the expression of a target RNA in a mammal, comprising administering to the mammal a pharmaceutical composition comprising the dsRNA of the present invention. Use of the dsRNA of the present invention for regulating the expression of a target RNA in a mammal. Use of the dsRNA of the present invention for producing a medicament for regulating the expression of a target RNA in a mammal.

[0307] The mammal is preferably a human. The route of administration is preferably enteral. In another embodiment, the route of administration is parenteral.

[0308] The 2'-O-XCE nucleotide according to the embodiment of the present invention can be produced with reference to WO 2007 / 102581, The Journal of Organic Chemistry, 2011, 76, pp 3042-3053, WO 2017 / 142054, Organic & Biomolecular Chemistry, 2019, 17, pp 4835-4842, etc.

[0309] Some embodiments of the present invention include the following: 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotides, 5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotides, and 5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotides.

[0310] 1. The following formula (II): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３ represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IIa): [In the formula, R １ and R ２are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４ each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], A １ is represented by the following formula (IIc): [Q １ and Q ２ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof.

[0311] 2. The following formula (III): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IIIa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IIIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], B １ is represented by the following formula (IIIc): [Q ３ and Q ４ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof.

[0312] 3. Formula (IV): wherein Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents, either alone or differently, selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group); and Z ３represents a hydrogen atom, a hydroxy-protecting group, or a phosphorus-containing group, X represents a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or a group represented by the following formula (IVa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (The R ３ and R ４each independently represent a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group). or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or the R ３ and R ４are taken together with the nitrogen atom to which they are bonded to form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group), n is an integer of 1 to 3, and when n is 2 or 3, then 2 or 3 R １ and R ２ may be the same or different, and T １ is represented by the following formula (IVb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group, or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group, or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), and Rb is an oxygen atom or a sulfur atom], E １ is represented by the following formula (IVc): [Q ５ ~Q ８ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group, or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group, and amino group are each independently unsubstituted or substituted with one or more substituents selected, either alone or differently, from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group, and a cyano group), or a salt thereof.

[0313] The 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide, 5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide, and 5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide according to this embodiment can be produced by the method shown below. However, the production method below shows an example of a general production method, and does not limit the method for producing the nucleotides according to this embodiment.

[0314] 5'-Vinylphosphonate (VP)-modified 2'-O-XCE nucleotide (3'-amidite derivative) can be synthesized, for example, by a method similar to that described in J. Med. Chem. 2018, 61, 734-744. Specifically, the 3'-hydroxy group of the 2'-O-XCE nucleotide is protected (e.g., with a tert-butyldimethylsilyl group), and then the 5'-hydroxy group is converted to an aldehyde by oxidation (e.g., with a Dess-Martin reagent), which is then converted to an olefin by a Horner-Wadsworth-Emmons reaction using tetrakis[(pivaloyloxy)methyl]methylenediphosphonate, followed by deprotection of the 3'-hydroxy-protecting group and further conversion to an amidite using an amidite-modifying reagent.

[0315] 5'-Cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide (3'-amidite derivative) can be synthesized, for example, by a method similar to that described in Chem. Commun., 2021, 57, 6808. Specifically, the hydroxy group at the 3' position of the 2'-O-XCE nucleotide is protected (for example, with a tert-butyldimethylsilyl group), and then the hydroxy group at the 5' position is converted to an aldehyde by oxidation (for example, with a Dess-Martin reagent), which is then converted to an olefin by a Wittig reaction or the like using O,O-diethyl P-[(triphenylphosphoranylidene)methyl]phosphonothioate, which is then converted to a cyclopropanated product by a Corey-Chaykovsky reaction or the like, and then the hydroxy-protecting group at the 3' position is deprotected, and then the thiophosphonate is converted to a phosphonate by oxidation (for example, with Oxone), which is then further converted to an amidite using an amiditizing reagent, thereby producing the nucleotide.

[0316] 5'-Ethylphosphonate (EP)-modified 2'-O-XCE nucleotide (3'-amidite) can be produced by reducing an olefin of a synthetic precursor of 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide (3'-amidite) by hydrogenation or the like, and then converting it into an amidite using an amidite-forming reagent.

[0317] The oligonucleotides according to this embodiment comprising a 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide, a 5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide, or a 5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide at the 5' end can be produced by solid-phase synthesis using an automated nucleic acid synthesizer (for example, nS-8II (Gene Design)) using the above-mentioned 3'-amidite derivatives and commercially available phosphoramidite reagents necessary for producing oligonucleotide analogs of desired nucleotide sequences.

[0318] Oligonucleotides containing a 5'-vinylphosphonate (VP)-modified 2'-O-XCE nucleotide and a 5'-ethylphosphonate (EP)-modified 2'-O-XCE nucleotide at the 5' end can be produced by treating the solid phase support after the reaction using the above-mentioned automated nucleic acid synthesizer with an amine (e.g., ammonia, methylamine, diethylamine, etc.).

[0319] An oligonucleotide containing a 5'-cyclopropanephosphonate (CPP)-modified 2'-O-XCE nucleotide at the 5' end can be produced by treating the solid phase support after the reaction using the above-mentioned automated nucleic acid synthesizer with, for example, a mixed solution of trimethylsilyl iodide, pyridine, and acetonitrile, and then treating it with an amine (e.g., ammonia, methylamine, diethylamine, etc.).

[0320] The present invention will be described in more detail below based on examples, but the scope of the present invention is not limited to these examples. Note that "NMR" means nuclear magnetic resonance, "LC / MS" means high performance liquid chromatography / mass spectrometry, "(v / v)" means (volume / volume), and "(w / v)" means (mass / volume). １When H NMR data is listed, it was measured at 400 MHz (JNM-ECZ400; JEOL Ltd.) or JNM-ECX300; JEOL Ltd.) and represents the chemical shift δ (unit: ppm) (splitting pattern, integral value) of the signal when tetramethylsilane was used as an internal standard. The chemical shift δ (unit: ppm) (splitting pattern, integral value) of the signal when tetramethylsilane was used as an internal standard is also shown. "s" stands for singlet, "d" stands for doublet, "t" stands for triplet, "dd" stands for doublet of doublets, "m" stands for multiplet, "br" stands for broad, "br s" stands for broad singlet, "J" stands for coupling constant, "CDCl3" stands for deuterated chloroform, "DMSO-D6" stands for deuterated dimethyl sulfoxide, and "ACETONITRILE-D3" stands for deuterated acetonitrile. ３１ When P-NMR data is given, it is measured at 162 MHz (JNM-ECZ400; manufactured by JEOL Ltd.) and the chemical shift δ (unit: ppm) of the signal is shown using phosphoric acid as an internal standard.

[0321] Unless otherwise specified, LC / MS was performed using the ESI (electrospray ionization) method under any of the following conditions 1 to 4. ＋ " means ESI positive ion mode, "ESI － " means ESI negative ion mode. The mixing ratio of the two solutions described below under LC / MS conditions is a volume ratio, and the content of formic acid is shown in volume %.

[0322] LC / MS Condition 1: Apparatus: Waters ACQUITY UPLC H CLASS / QDa Detector Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient conditions: After starting the measurement at a flow rate of 0.6 mL / min and a mixing ratio of Solution A to Solution B of 90 / 10, the mixing ratio of Solution A to Solution B was linearly changed to 10 / 90 over 3 minutes. The mixing ratio of Solution A to Solution B was then fixed at 10 / 90 for 0.7 minutes. The mixing ratio of Solution A to Solution B was then linearly changed to 90 / 10 over 0.1 minutes at a flow rate of 0.8 mL / min. The mixing ratio of Solution A to Solution B was then fixed at 90 / 10 for 1 minute. Detection wavelength: PDA (190-400 nm)

[0323] LC / MS Condition 2: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 0.1% formic acid aqueous solution Solution B: 0.1% formic acid-acetonitrile solution Gradient conditions: After starting the measurement at a flow rate of 0.6 mL / min and a mixing ratio of Solution A to Solution B of 90 / 10, the mixing ratio of Solution A to Solution B was linearly changed to 10 / 90 over 3 minutes. The mixing ratio of Solution A to Solution B was then fixed at 10 / 90 for 0.7 minutes. The mixing ratio of Solution A to Solution B was then linearly changed to 90 / 10 over 0.1 minutes at a flow rate of 0.8 mL / min. The mixing ratio of Solution A to Solution B was then fixed at 90 / 10 for 1 minute. Detection wavelength: PDA (190-400 nm)

[0324] LC / MS Condition 3: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 10 mmol / L ammonium bicarbonate aqueous solution Solution B: 10 mmol / L ammonium bicarbonate-acetonitrile solution (containing 10% of the total solution volume of water) Gradient conditions: After starting the measurement at a flow rate of 0.6 mL / min and a mixing ratio of Solution A to Solution B of 91 / 9, the mixing ratio of Solution A to Solution B was linearly changed to 19 / 81 over 3 minutes. The mixing ratio of Solution A to Solution B was then fixed at 19 / 81 for 0.7 minutes. The mixing ratio of Solution A to Solution B was then linearly changed to 91 / 9 over 0.1 minutes at a flow rate of 0.8 mL / min. After that, the mixing ratio of solution A and solution B was fixed at 91 / 9 for 1 minute. Detection wavelength: PDA (190-400 nm).

[0325] LC / MS Condition 4: Apparatus: Waters ACQUITY UPLC H CLASS / SQ Detector 2 Column: Waters ACQUITY UPLC BEH C18 (1.7 μm, 2.1 × 50 mm) Column temperature: 40°C Solvent: Solution A: 10 mmol / L ammonium bicarbonate aqueous solution Solution B: 10 mmol / L ammonium bicarbonate-acetonitrile solution (containing 10% water by total solution volume) Gradient conditions: After starting the measurement at a flow rate of 0.6 mL / min and a mixing ratio of Solution A to Solution B of 82 / 18, the mixing ratio of Solution A to Solution B was linearly changed to 10 / 90 over 2.5 minutes. After that, the mixing ratio of Solution A to Solution B was fixed at 19 / 81 for 1.2 minutes. After that, the mixing ratio of Solution A to Solution B was linearly changed to 82 / 18 over 0.1 minutes at a flow rate of 0.8 mL / min. After that, the mixing ratio of solution A and solution B was fixed at 82 / 18 for 1 minute. Detection wavelength: PDA (190-400 nm).

[0326] For purification by silica gel column chromatography, unless otherwise specified, Purif-Pack (registered trademark)-EX (SI-50 μm) manufactured by SHOKO SCIENCE was used. For purification by silica gel reverse phase column chromatography, unless otherwise specified, either Xbridge PREP C18 5 μm 19 x 100 mm or Xselect CSH Prep C18 5 μm 19 x 100 mm was used. The contents and amounts of formic acid and triethylamine added to the solvent used in silica gel column chromatography are expressed in volume %.

[0327] Purification by supercritical fluid chromatography was carried out under the following conditions: Apparatus: Waters Prep 80q Column: DAICEL CHIRALPAK IG (20 mmφ×250 mm, 5 μm) Column temperature: 40° C. Mobile phase: carbon dioxide / methanol = 50 / 50, Flow rate: 15 mL / min

[0328] The single crystal X-ray structural analysis was carried out using a single crystal X-ray diffractometer, XtaLAB Synergy R, DW system, Hypix (manufactured by Rigaku Corporation), in accordance with the instruction manual.

[0329] [Production Example 1] Synthesis of Compound 8

[0330] Step (1): Synthesis of Compound 2. Imidazole (947.7 mg, 13.9 mmol) was added to a mixture of compound 1 (synthesized according to the method described in International Publication No. WO 2023 / 054708) (3.00 g, 4.64 mmol) and dehydrated N,N-dimethylformamide (9.30 mL), and the mixture was stirred at room temperature to dissolve. tert-Butyldimethylchlorosilane (1.40 g, 9.28 mmol) was added to the mixture, and the mixture was stirred overnight (approximately 16-20 hours) at room temperature under an argon atmosphere. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with water and a saturated aqueous solution of sodium chloride, dried over sodium sulfate, filtered, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain the desired compound 2 (3.47 g, 98% yield) as a white solid. LC / MS: Condition 1 LC / MS (ESI-) m / z; 758.1 [MH] －　 1 H-NMR (CDCl3) δ: 8.20 (1H, br s), 7.78-7.77 (1H, m), 7.40-7.38 (2H, m), 7.32-7.28 (7H, m), 6.85-6.83 (4H, m), 6.35-6.34 (1H, m), 5.95 (1H, d, J = 3.3 Hz), 4.34-4.32 (1H, m), 4.10-4.07 (1H, m), 3.95-3.89 (3H, m), 3.80 (6H, s), 3.66 (1H, dd, J = 11.0, 2.2 Hz), 3.26 (1H, dd, J = 11.0, 2.6 Hz), 2.78 (3H, d, J = 4.8 Hz), 2.52-2.48 (2H, m), 1.37-1.37 (3H, m), 0.80 (9H, s), 0.04 (3H, s), -0.07 (3H, s).

[0331] Step (2): Synthesis of Compound 3 To a mixture of compound 2 (3.47 g, 4.57 mmol) and methylene chloride (44.0 mL), 1-dodecanethiol (2.17 mL, 9.13 mmol) was added, followed by trifluoroacetic acid (0.525 mL, 6.85 mmol), and the mixture was stirred at room temperature for 15 minutes under an argon atmosphere. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture until the pH changed to 8-9, and the mixture was extracted with methylene chloride. The resulting organic layer was washed with a saturated aqueous solution of sodium chloride, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to obtain the desired compound 3 (1.88 g, 90%) as a white solid. LC / MS: Condition 1 LC / MS (ESI+) m / z: 458.4 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.44 (1H, br s), 7.55-7.55 (1H, m), 6.22-6.20 (1H, m), 5.70 (1H, d, J = 4.0 Hz), 4.37 (1H, t, J = 5.1 Hz), 4.08-4.03 (2H, m), 4.02-3.98 (1H, m), 3.92-3.90 (1H, m), 3.85-3.74 (2H, m), 2.78 (3H, d, J = 4.8 Hz), 2.54-2.52 (1H, m), 2.48-2.46 (2H, m), 1.93 (3H, d, J = 1.1 Hz), 0.92 (9H, s), 0.12 (6H, s).

[0332] Step (3): Synthesis of Compound 6 To a mixture of compound 3 (500.0 mg, 1.1 mmol) and methylene chloride (25.0 mL) was added 1,1,1-triacetoxy-1,1-dihydro-1,2-benziodoxol-3-(1H)-one (Dess-Martin periodinane) (648.8 mg, 1.5 mmol) at 0°C, and the mixture was stirred at 0°C for 1 hour, followed by further stirring at room temperature for 1 hour. After the reaction, a mixed solution of 10% (w / v) aqueous sodium thiosulfate solution (10.8 mL) and saturated aqueous sodium bicarbonate solution (10.8 mL) was added at 0°C, and the mixture was extracted with ethyl acetate. The resulting organic layer was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product containing the desired compound 4. Next, tetrahydrofuran (2.5 mL) was added to sodium hydride (55 wt % liquid paraffin, 182.1 mg, 3.8 mmol), and compound 5 was added dropwise at −78°C under an argon atmosphere. The mixture was stirred at −78°C for 15 minutes. Subsequently, a tetrahydrofuran solution (6.9 mL) of the crude product containing compound 4 was added dropwise to the reaction mixture. The mixture was stirred at −78°C for 1 hour, then warmed to 0°C and stirred for 1 hour, and then warmed to room temperature and stirred for 4 hours. After the reaction, saturated aqueous ammonium chloride solution was added, and the mixture was extracted with ethyl acetate. The resulting organic layer was washed with saturated aqueous sodium chloride solution, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to obtain the desired compound 6 (713.0 mg, two-step yield: 86%) as a colorless, transparent oil. LC / MS: Condition 1 LC / MS (ESI-) m / z; 760.4 [MH] － 1H-NMR (400 MHz, CDCl3) δ: 0.10 (s, 6H), 0.88-0.94 (m, 9H), 1.21-1.23 (m, 18H), 1.94-1.98 (m, 3H), 2.45-2.49 (m, 2H), 2.79-2.82 (m, 3H), 3.81-3.86 (m, 1H), 3.89-3.95 (m, 2H), 4.06-4.10 (m, 1H), 4.44-4.48 (m, 1H), 5.63-5.72 (m, 4H), 5.82 (d, J = 3.3 Hz, 1H), 6.07-6.09 (m, 1H), 6.81-6.92 (m, 1H), 7.12-7.12 (m, 1H), 8.01 (s, 1H). 31 P-NMR (162 MHz, CDCl3) δ: 14.3, 16.9.

[0333] Step (4): Synthesis of Compound 7 Compound 6 (713.0 g, 0.94 mmol) was added with a formic acid / water mixture (35.7 mL, 1 / 1 (v / v)) and stirred at room temperature for 8 hours. To this reaction mixture, a formic acid / water mixture (35.7 mL, 1 / 1 (v / v)) was added and stirred at room temperature for 13 hours. Methanol was added to the reaction mixture, which was then stirred and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol) to obtain a crude product containing the desired compound 7. This crude product was purified by silica gel reverse-phase column chromatography (0.1% trifluoroacetic acid-acetonitrile / 0.1% aqueous trifluoroacetic acid solution) to obtain the desired compound 7 (400.1 mg, 66% yield) as a colorless, transparent oil. LC / MS: Condition 4 LC / MS (ESI+) m / z: 648.7 [M+H] ＋ 1 ​H NMR (400 MHz, DMSO-D6) δ: 0.97-1.17 (m, 18H), 2.06 (s, 3H), 2.31-2.36 (m, 2H), 2.54 (d, 3H), 3.64-3.79 (m, 3H), 4.10-4.19 (m, 2H), 4.30-4.33 (m, 1H), 5.58-5.62 (m, 4H), 5.78 (d, 1H), 6.02-6.12 (m, 1H), 6.77-6.89 (m, 1H), 7.44-7.44 (m, 1H), 7.84-7.85 (m, 1H), 11.40 (s, 1H) 31 P-NMR (162 MHz, DMSO-D6) δ: 17.9

[0334] Step (5): Synthesis of Compound 8 To a mixture of compound 7 (400.1 mg, 0.62 mmol) and acetonitrile (8.0 mL), 4,5-dicyanoimidazole (80.3 mg, 0.68 mmol) and 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (235.2 μL, 0.74 mmol) were added and stirred at room temperature for 2 hours. To the reaction mixture, 1.5% aqueous potassium bicarbonate solution (8.0 mL) was added. After stirring, hexane (2.0 mL) and ethyl acetate (2.0 mL) were added. After stirring for 10 minutes, the aqueous layer was removed. Next, 1.5% aqueous potassium bicarbonate solution (8.0 mL) was added to the organic layer. After stirring for 10 minutes, the aqueous layer was removed. This step was repeated once more, and water and saturated aqueous sodium chloride solution were added to the organic layer. After stirring, the aqueous layer was removed, and the organic layer was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (ethyl acetate (0.15% triethylamine added) / methanol) to obtain the target compound 8 (197.7 mg, yield 38%) as a white solid. LC / MS: Condition 3 LC / MS (ESI-) m / z; 846.3, 846.2 [M-H] －　 1H-NMR (400 MHz, ACETONITRILE-D3) δ: 1.17-1.23 (m, 30H), 1.82-1.83 (m, 2H), 2.07-2.10 (m, 3H), 2.28-2.36 (m, 2H), 2.62 (t, 3H), 2.66-2.76 (m, 2H), 3.59-3.89 (m, 4H), 4.09-4.13 (m, 1H), 4.24-4.37 (m, 1H), 4.51-4.58 (m, 1H), 5.57-5.65 (m, 4H), 5.82-5.84 (m, 1H), 6.04-6.14 (m, 1H), 6.32-6.35 (m, 1H), 6.78-7.00 (m, 1H), 7.16-7.17 (m, 1H). 31 P-NMR (162 MHz, ACETONITRILE-D3) δ: 150.4, 150.3, 17.3, 16.9.

[0335] [Production Example 2] Synthesis of Compound 14

[0336] Step (1): Synthesis of Compound 10 A mixture of compound 3 (1.06 g, 2.32 mmol) and methylene chloride (46.5 mL) was cooled to 0°C, and 1,1,1-triacetoxy-1,1-dihydro-1,2-benzoiodoxol-3-(1H)-one (Dess-Martin periodinane) (1.18 g, 2.79 mmol) and saturated sodium bicarbonate (19.5 mg, 0.23 mmol) were added. The mixture was stirred under an argon atmosphere at 0°C for 1 hour and then at room temperature for 30 minutes. To the reaction mixture was added a mixed solution of saturated aqueous sodium bicarbonate and saturated aqueous sodium thiosulfate (40.0 mL, 1 / 1 (v / v)), and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product (1.08 g) of the desired compound 4 as a white solid. This crude product (1.07 mmol) was dissolved in dehydrated tetrahydrofuran (8.0 mL), and a tetrahydrofuran solution (6.0 mL) of compound 9 (synthesized according to the method described in Chem. Commun., 2021, 57, 6808) (1.19 g, 2.79 mmol) was added dropwise at 0 °C under an argon atmosphere. The mixture was then warmed to room temperature and stirred overnight (approximately 16 to 20 hours). The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate → ethyl acetate / methanol) to obtain the desired compound 10 (976.7 mg, two-step yield 69%) as a white solid. LC / MS: Condition 1 LC / MS (ESI+) m / z: 606.2 [M+H] ＋ 1H-NMR (CDCl3) δ: 8.78 (1H, br s), 7.14-7.13 (1H, m), 6.86-6.78 (1H, m), 6.28-6.22 (2H, m), 5.82 (1H, d, J = 2.5 Hz), 4.55-4.49 (1H, m), 4.19-3.93 (6H, m), 3.88-3.86 (2H, m), 2.80-2.79 (3H, m), 2.53-2.45 (2H, m), 1.96-1.96 (3H, m), 1.32 (6H, td, J = 7.2, 1.6 Hz), 0.92 (9H, s), 0.12 (6H, d, J = 3.7 Hz). 31 P-NMR (162 MHz, CDCl3) δ: 84.4

[0337] Step (2): Synthesis of Compound 12. Under an argon atmosphere, sodium hydride (55 wt % liquid paraffin, 112.9 mg, 4.7 mmol) was added to a mixture of trimethylsulfoxonium iodide (1.04 g, 4.7 mmol) and dehydrated dimethyl sulfoxide (4.0 mL) and stirred at room temperature for 30 minutes. This suspension was added dropwise to a dimethyl sulfoxide solution (3 mL) of compound 10 (949.8 mg, 1.57 mmol). After the addition was complete, the mixture was stirred at room temperature for 90 minutes and then heated and stirred at 40°C for 6 hours. The reaction mixture was cooled to 0°C, ice water was added, and the mixture was extracted with ethyl acetate. The organic layer was washed with water and saturated aqueous sodium chloride, dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product of the desired compound 11 as a white solid. Subsequently, a mixed solution of formic acid / water (10.08 mL, 1 / 1 (v / v)) was added to the crude product of compound 11 (937.1 mg), and the mixture was stirred overnight (approximately 16 to 20 hours) at room temperature. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol) to obtain the target compound 12, isomer A (455.0 mg, LC / MS (condition 1) RT = 1.69 min, white solid, yield 60%) and isomer B (204.5 mg, LC / MS (condition 1) RT = 1.74 min, white solid, yield 27%). Isomer A LC / MS: Condition 1 LC / MS (ESI+) m / z: 506.3 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.64 (1H, br s), 7.23-7.22 (1H, m), 5.88-5.86 (2H, m), 4.23-4.21 (1H, m), 4.19-4.03 (4H, m), 4.00-3.97 (2H, m), 3.86-3.80 (1H, m), 3.51-3.48 (1H, m), 2.82 (3H, d, J = 4.5 Hz), 2.60-2.50 (1H, m), 2.45-2.40 (1H, m), 1.96-1.96 (3H, m), 1.66-1.51 (2H, m), 1.36-1.24 (7H, m), 1.23-1.15 (1H, m), 1.11-1.00 (1H, m). 31P-NMR (162 MHz, CDCl3) δ 101.4 Isomer B LC / MS: Condition 1 LC / MS (ESI+) m / z; 506.3 [M+H] ＋ 1 H-NMR (CDCl3) δ: 8.74 (1H, s), 7.16 (1H, d, J = 1.2 Hz), 5.98-5.96 (1H, m), 5.77 (1H, d, J = 3.7 Hz), 4.25-3.94 (7H, m), 3.88-3.82 (1H, m), 3.54-3.47 (1H, m), 2.82 (3H, d, J = 4.9 Hz), 2.60-2.53 (1H, m), 2.45-2.40 (1H, m), 1.96-1.96 (3H, m), 1.65-1.50 (1H, m), 1.36-1.29 (7H, m), 1.28-1.18 (1H, m), 1.04-0.98 (1H, m). 31 P-NMR (162 MHz, CDCl3) δ: 101.0

[0338] Step (3): Synthesis of Isomer A of Compound 13. Compound 12 (Isomer A: 455.0 mg, 0.9 mmol) was dissolved in a tetrahydrofuran / water mixture (6.5 mL, 1 / 1 (v / v)), and OXONE® monopersulfated compound (553.3 mg, 1.8 mmol) was added. The mixture was stirred at room temperature for 5 hours. Ethanol was added to the reaction mixture, which was then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel reverse-phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to obtain the desired Isomer A of Compound 13 (102.25 mg, LC / MS (Condition 1) RT = 1.14 min, 23% yield) as a white solid. LC / MS: Condition 1 LC / MS (ESI+) m / z: 490.2 [M+H] ＋ 1H-NMR (CDCl3) δ: 9.43 (1H, br s), 7.22-7.22 (1H, m), 6.32-6.31 (1H, m), 5.82 (1H, d, J = 4.5 Hz), 4.70 (1H, br s), 4.24-4.22 (1H, m), 4.16-4.05 (4H, m), 4.00-3.97 (2H, m), 3.85-3.79 (1H, m), 3.43-3.40 (1H, m), 2.80 (3H, d, J = 4.5 Hz), 2.62-2.39 (2H, m), 1.93 (3H, s), 1.71-1.57 (1H, m), 1.34-1.29 (6H, m), 1.24-1.18 (1H, m), 1.09-0.92 (2H, m). 31 P-NMR (162 MHz, CDCl3) δ: 29.6

[0339] Step (3): Synthesis of Isomer A of Compound 13 (Alternative Method) This is another example of the preparation of Isomer A of Compound 13. Compound 12 (Isomer A: 485.3 mg, 0.96 mmol) was dissolved in dichloromethane (33 mL), and 3-chloroperbenzoic acid (containing approximately 30% water by weight) (1.08 g, 1.63 mmol) was added at 0°C. The mixture was then warmed to room temperature and stirred for 15 minutes. A mixed solution of 30% (w / v) aqueous sodium thiosulfate and saturated aqueous sodium bicarbonate (1 / 1 (v / v)) was then added to the reaction mixture, and the mixture was stirred at room temperature for 5 minutes. The mixed solution was concentrated under reduced pressure, and ethanol and methanol were added to the resulting residue. The precipitated solid was filtered, and the filtrate was collected in a recovery flask. Ethanol was added to the collected solid, and the solid was filtered. The filtrates obtained from the first and second filtrations were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel reverse phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to obtain the target isomer A of Compound 13 (241.2 mg, yield 51%) as a white solid.

[0340] 1 mg of isomer A of compound 13 obtained in the above step (3) was diluted with 50 μL of acetone and allowed to stand at 25° C. for 24 hours to obtain crystals.

[0341] To determine the crystal structure, single-crystal X-ray analysis was performed. The diffraction data was analyzed using the software CrysAlisPro (Rigaku), after which integration and absorption correction were performed. The initial structure was obtained using SHELX T (Acta Crystallogr. Sect. A 2015, 71, 3-8), and refined using SHELX L (Acta Crystallogr. Sect. C 2015, 71, 3-8). The results are shown in Table 1, and the ORTEP diagram is shown in Figure 1.

[0342]

[0343] From the above results, the structure of Isomer A of Compound 13 was determined to be the following formula (13A).

[0344] The structure of isomer B of compound 13 was determined to be the following formula (13B).

[0345] Step (3'): Synthesis of Isomer B of Compound 13. Compound 12 (Isomer B: 204.5 mg, 0.40 mmol) was dissolved in a tetrahydrofuran / water mixture (3.2 mL, 1 / 1 (v / v)), and OXONE® monopersulfated compound (248.1 mg, 0.81 mmol) was added. The mixture was stirred at room temperature for 5 hours. Ethanol was added to the reaction mixture, which was then filtered. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel reverse-phase column chromatography (0.1% formic acid-acetonitrile / 0.1% formic acid aqueous solution) to obtain the desired Isomer B of Compound 13 (76.60 mg, LC / MS (Condition 1) RT = 1.17 min, 39% yield) as a colorless, transparent oil. LC / MS: Condition 1 LC / MS (ESI+) m / z: 490.3 [M+H] ＋ 1H-NMR (CDCl3) δ: 8.90 (1H, br s), 7.17-7.17 (1H, m), 6.07-6.06 (1H, m), 5.77 (1H, d, J = 3.7 Hz), 4.20-4.07 (5H, m), 4.02-3.97 (2H, m), 3.88-3.82 (1H, m), 3.42 (1H, dd, J = 8.4, 5.9 Hz), 2.82 (3H, d, J = 4.9 Hz), 2.59-2.54 (1H, m), 2.45-2.40 (1H, m), 1.95-1.95 (3H, m), 1.68-1.55 (1H, m), 1.35 (6H, td, J = 7.0, 2.0 Hz), 1.29-1.17 (1H, m), 1.08-0.92 (2H, m). 31 P-NMR (162 MHz, CDCl3) δ: 29.7

[0346] Step (3'): Synthesis of Isomer B of Compound 13 (Alternative Method) This is another example of the preparation of Isomer B of Compound 13. Compound 12 (Isomer B: 1.83 g, 3.62 mmol) was dissolved in dichloromethane (124 mL), and 3-chloroperbenzoic acid (containing approximately 30% w...

Claims

1. A double-stranded RNA capable of inhibiting the expression of a target RNA, wherein the double-stranded RNA comprises a sense strand and an antisense strand, the antisense strand has 14 to 40 nucleotides and has complementarity with the target RNA sufficient to mediate RNA interference, the sense strand has 14 to 40 nucleotides and has complementarity with the antisense strand, and the double-stranded RNA comprises at least one 2'-O-XCE nucleotide.

2. The double-stranded RNA according to claim 1, wherein the antisense strand comprises at least one 2'-O-XCE nucleotide or the sense strand comprises at least one 2'-O-XCE nucleotide.

3. The double-stranded RNA according to claim 1 or 2, wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-fluoro nucleotide.

4. The double-stranded RNA according to any one of claims 1 to 3, wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one 2'-O-Me nucleotide.

5. The double-stranded RNA according to any one of claims 1 to 4, wherein the double-stranded RNA comprises at least one 2'-O-XCE nucleotide and at least one deoxyribonucleotide.

6. The double-stranded RNA according to any one of claims 1 to 5, wherein the antisense strand comprises a 2'-O-XCE nucleotide at at least one of positions 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22 and 23 counted from the 5'-end of the antisense strand hybridizing portion.

7. The double-stranded RNA according to any one of claims 1 to 6, wherein the sense strand comprises a 2'-O-XCE nucleotide at at least one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22 and 23 counted from the 3'-end of the sense strand hybridizing portion.

8. The 2′-O-XCE nucleotide is represented by the following formula (I): {In the formula, Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group, or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group, and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents selected singly or differently from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group, and a protected sulfanyl group), X is a hydrogen atom, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected singly or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), or the following formula (Ia): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group, or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents selected singly or differently from the group consisting of a halogen atom, a C1-6 alkoxy group, and a cyano group), Y is NR ３ R ４ (the R ３ and R ４ is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group, and are each independently substituted with one or more substituents selected from the group consisting of these), or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group, and are each independently substituted with one or more substituents selected from the group consisting of these), or the R ３ and R ４ forms, together with the nitrogen atom to which they are attached, a 3- to 11-membered nitrogen-containing non-aromatic heterocyclic ring (the 3- to 11-membered nitrogen-containing non-aromatic heterocyclic ring is unsubstituted or is substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group)), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or is substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group)), n is an integer from 1 to 3, and when n is 2 or 3, two or three Rs １ and Rs ２ may be the same or different from each other], and is a nucleotide containing the partial structure represented by}, the double-stranded RNA according to any one of claims 1 to 7.

9. The double-stranded RNA according to claim 8, wherein X is a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group).

10. The double-stranded RNA according to claim 8 or 9, wherein X is a methyl group.

11. X is represented by the following formula (Ia): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), Y is NR ３ R ４ (the R ３ and R ４ are each independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group) or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group)), or the R ３ and R ４ forms, together with the nitrogen atom to which they are attached, a 3- to 11-membered nitrogen-containing non-aromatic heterocyclic ring (the 3- to 11-membered nitrogen-containing non-aromatic heterocyclic ring is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), n is an integer from 1 to 3, and when n is 2 or 3, two or three Rs １ and Rs ２ may be the same or different from each other], and the double-stranded RNA according to claim 8.

12. The said R １ and R ２ are hydrogen atoms, and the double-stranded RNA according to claim 11.

13. wherein Y is NR ３ R ４ and the R ３ and R ４ together with the nitrogen atom to which they are attached form a ring containing 4 to 6 methylene groups among 4- to 8-membered nitrogen-containing non-aromatic heterocycles, or Y is a C2-9 aromatic heterocyclic group, the double-stranded RNA according to claim 11 or 12.

14. wherein Y is NR ３ R ４ and R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, the double-stranded RNA according to any one of claims 11 to 13.

15. The double-stranded RNA according to any one of claims 11 to 13, wherein Y is a pyridyl group, an imidazolyl group or a benzimidazolyl group.

16. The double-stranded RNA according to any one of claims 11 to 15, wherein n is 2.

17. The double-stranded RNA according to any one of claims 1 to 16, wherein the antisense strand contains 1, 2, 3, 4, 5 or 6 2'-fluoronucleotides.

18. The double-stranded RNA according to any one of claims 1 to 17, wherein the sense strand contains 1, 2, 3, 4 or 5 2'-fluoronucleotides.

19. The double-stranded RNA according to any one of claims 1 to 18, wherein the sense strand contains 7 to 18 2'-O-Me nucleotides.

20. The double-stranded RNA according to any one of claims 1 to 19, wherein the antisense strand contains 13 to 20 2'-O-Me nucleotides.

21. The double-stranded RNA according to any one of claims 1 to 20, wherein the sense strand contains 0 to 6 deoxyribonucleotides.

22. The double-stranded RNA according to any one of claims 1 to 21, wherein the antisense strand contains 0 to 6 deoxyribonucleotides.

23. The double-stranded RNA according to any one of claims 1 to 22, wherein the double-stranded RNA contains at least one phosphorothioate bond.

24. The double-stranded RNA according to any one of claims 1 to 23, wherein the sense strand contains 1, 2, 3 or 4 phosphorothioate bonds.

25. The double-stranded RNA according to any one of claims 1 to 24, wherein the sense strand contains a phosphorothioate bond at at least one selected from the group consisting of between nucleotide positions 1 and 2, between 2 and 3, counting from the 5'-end of the sense strand, between nucleotide positions 1 and 2 and between 2 and 3, counting from the 3'-end.

26. The double-stranded RNA according to any one of claims 1 to 25, wherein the antisense strand contains 1, 2, 3 or 4 phosphorothioate bonds.

27. The antisense strand contains phosphorothioate linkages between nucleotide positions 1 and 2, 2 and 3, counting from the 5'-end of the antisense strand, and between nucleotide positions 1 and 2, and 2 and 3, counting from the 3'-end of the antisense strand, of the double-stranded RNA according to any one of claims 1 to 26.

28. The antisense strand contains a 5'-phosphonate group at the 5'-end of the antisense strand, of the double-stranded RNA according to any one of claims 1 to 27.

29. The double-stranded RNA according to claim 28, wherein the 5'-phosphonate group is a 5'-vinylphosphonate group.

30. The antisense strand contains a 5'-vinylphosphonylated 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 29.

31. The antisense strand contains a 5'-vinylphosphonylated 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 29.

32. The double-stranded RNA according to claim 28, wherein the 5'-phosphonate group is a 5'-cyclopropylphosphonate group.

33. The antisense strand contains a 5'-cyclopropylphosphonylated 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 32.

34. The antisense strand contains a 5'-cyclopropylphosphonylated 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 32.

35. The double-stranded RNA according to claim 28, wherein the 5'-phosphonate group is a 5'-ethylphosphonate group.

36. The antisense strand contains a 5'-ethylphosphonylated 2'-O-Me nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 35.

37. The antisense strand contains a 5'-ethylphosphonylated 2'-O-XCE nucleotide at position 1, counting from the 5'-end of the antisense strand, of the double-stranded RNA according to claim 28 or 35.

38. The nucleotide lengths of the sense strand and the antisense strand are independently 19 to 25, of the double-stranded RNA according to any one of claims 1 to 37.

39. The double-stranded RNA according to any one of claims 1 to 38, wherein the nucleotide length of the sense strand is 21.

40. The double-stranded RNA according to any one of claims 1 to 39, wherein the nucleotide length of the antisense strand is 23.

41. The double-stranded RNA according to any one of claims 1 to 40, wherein the nucleotide length of the sense strand is 21 and the nucleotide length of the antisense strand is 23.

42. The double-stranded RNA according to any one of claims 1 to 41, wherein the antisense strand contains an overhang at the 3' end.

43. The double-stranded RNA according to any one of claims 1 to 42, wherein the antisense strand contains a blunt end at the 5' end.

44. The double-stranded RNA according to any one of claims 1 to 43, further comprising a group derived from a functional molecule having at least one function selected from the group consisting of a labeling function, a purification function, and a delivery function to a target site.

45. The double-stranded RNA according to claim 44, wherein the functional molecule is selected from the group consisting of sugars, lipids, peptides, proteins, and derivatives thereof.

46. The double-stranded RNA according to claim 44 or 45, wherein the functional molecule is selected from the group consisting of cholesterol, vitamins, steroids, C5-30 saturated fatty acids, C5-30 unsaturated fatty acids, C5-30 alkyl groups, and C5-30 alkenyl groups.

47. The double-stranded RNA according to claim 44 or 45, wherein the functional molecule is a peptide or protein selected from the group consisting of a ligand of a receptor and an antibody.

48. The double-stranded RNA according to claim 44 or 45, wherein the functional molecule is a sugar derivative that interacts with an asialoglycoprotein receptor.

49. A pharmaceutical composition comprising the double-stranded RNA according to any one of claims 1 to 48 and a pharmaceutically acceptable carrier.

50. A method for controlling the function of a target RNA, comprising the step of contacting the double-stranded RNA according to any one of claims 1 to 48 with a cell.

51. A method for controlling the function of a target RNA in a mammal, comprising the step of administering the pharmaceutical composition according to claim 49 to the mammal.

52. A method for producing the double-stranded RNA according to any one of claims 1 to 48 using 2'-O-XCE nucleotides.

53. The following formula (II): {In the formula, Base is a purin-9-yl group, 2-oxo-pyrimidin-1-yl group or 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, 2-oxo-pyrimidin-1-yl group and 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group), and Z ３ is a hydrogen atom, a hydroxy protecting group or a phosphorus-containing group, X is a hydrogen atom, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), or the following formula (IIa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (the R ３ and R ４ is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group, which are selected individually or differently from the group consisting of one or more substituents), or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group, which are selected individually or differently from the group consisting of one or more substituents), or the R ３ and R ４ forms, together with the nitrogen atom to which they are attached, a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), n is an integer from 1 to 3, and when n is 2 or 3, two or three Rs １ and Rs ２ may be the same or different from each other], and T １ is represented by the following formula (IIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group), and Rb is an oxygen atom or a sulfur atom], and A １ is represented by the following formula (IIc): [Q １ and Q ２ are each independently selected from a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group and amino group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group)], or a salt thereof.

54. The X is a methyl group, and Q １ and Q ２ are hydrogen atoms, the compound according to claim 53, or a salt thereof.

55. Said R １ and R ２ are hydrogen atoms, said Y is NR ３ R ４ wherein said R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, and Q １ and Q ２ are hydrogen atoms, a compound according to claim 53 or 54, or a salt thereof.

56. The phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group, the compound according to any one of claims 53 to 55, or a salt thereof.

57. The following formula (III): {In the formula, Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or are substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group), and Z ３ is a hydrogen atom, a hydroxy protecting group or a phosphorus-containing group, X is a hydrogen atom, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or are substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), or the following formula (IIIa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or are substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (the R ３ and R ４ is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or are substituted with one or more substituents independently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or is substituted with one or more substituents independently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group) or the R ３ and R ４ together with the nitrogen atom to which they are attached, form a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group)), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group)), n is an integer from 1 to 3, and when n is 2 or 3, two or three Rs １ and Rs ２ may be the same or different from each other], and T １ is represented by the following formula (IIIb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group), and Rb is an oxygen atom or a sulfur atom] and is a group represented by, B １ is the following formula (IIIc): [Q ３ and Q ４ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group and amino group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group)] and is a group represented by}, or a salt thereof.

58. X is a methyl group, Q ３ and Q ４ are hydrogen atoms, the compound according to claim 57, or a salt thereof.

59. The R １ and R ２ are hydrogen atoms, the Y is NR ３ R ４ wherein the R ３ and R ４ together with the nitrogen atom to which they are attached form morpholine, Q ３ and Q ４ are hydrogen atoms, the compound according to claim 57 or 58, or a salt thereof.

60. The phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group, the compound according to any one of claims 57 to 59, or a salt thereof.

61. The following formula (IV): {In the formula, Base is a purin-9-yl group, a 2-oxo-pyrimidin-1-yl group or a 2-thioxo-pyrimidin-1-yl group (the purin-9-yl group, the 2-oxo-pyrimidin-1-yl group and the 2-thioxo-pyrimidin-1-yl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, an amino group, a protected amino group, a hydroxy group, a protected hydroxy group, a sulfanyl group and a protected sulfanyl group), and Z ３ is a hydrogen atom, a hydroxy protecting group or a phosphorus-containing group, and X is a hydrogen atom, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), or the following formula (IVa): [In the formula, R １ and R ２ are each independently a hydrogen atom, a halogen atom, a cyano group, a C1-6 alkyl group or a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkoxy group and a cyano group), and Y is NR ３ R ４ (the R ３ and R ４ is independently a hydrogen atom, a C1-6 alkyl group, a C2-6 alkenyl group (the C1-6 alkyl group and the C2-6 alkenyl group are each independently unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group, which are selected singly or differently from the group consisting of one or more substituents), or a C7-10 aralkyl group (the C7-10 aralkyl group is unsubstituted or a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group and a C1-6 alkoxycarbonylamino group, which are selected singly or differently from the group consisting of one or more substituents), or the R ３ and R ４ forms, together with the nitrogen atom to which they are attached, a 3- to 11-membered nitrogen-containing non-aromatic heterocycle (the 3- to 11-membered nitrogen-containing non-aromatic heterocycle is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), or a C2-9 aromatic heterocyclic group (the C2-9 aromatic heterocyclic group is unsubstituted or substituted with one or more substituents independently or differently selected from the group consisting of a halogen atom, a cyano group, a nitro group, an amino group, a hydroxy group, a carboxy group, a carbamoyl group, a C1-6 alkyl group, a C2-6 alkenyl group, a C1-6 alkoxy group, a C2-6 alkenyloxy group, a C1-6 alkoxycarbonyl group, a C2-6 alkenyloxycarbonyl group, a C1-6 alkylcarbonyl group, a C1-6 haloalkyl group, a C1-6 alkylamino group, a C1-6 alkylaminocarbonyl group, a C1-6 alkylcarbonyloxy group, a C1-6 alkylcarbonylamino group, and a C1-6 alkoxycarbonylamino group)), n is an integer from 1 to 3, and when n is 2 or 3, two or three Rs １ and Rs ２ may be the same or different from each other], and T １ is represented by the following formula (IVb): [Ra and Rc are each independently selected from a hydroxy group, a protected hydroxy group, a thiol group, a protected thiol group, an amino group, a protected amino group, a C1-6 alkyl group, a C2-6 alkenyl group or a C1-6 alkoxy group (the hydroxy group, thiol group, amino group, C1-6 alkyl group, C2-6 alkenyl group or C1-6 alkoxy group is each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group), and Rb is an oxygen atom or a sulfur atom] and is a group represented by, E １ is the following formula (IVc): [Q ５ ~Q ８ are each independently a hydrogen atom, a halogen atom, a C1-C6 alkyl group, a C1-6 alkoxy group, a C2-6 alkenyl group, a C2-6 alkynyl group or an amino group (the C1-C6 alkyl group, C1-6 alkoxy group, C2-6 alkenyl group, C2-6 alkynyl group and amino group are each independently unsubstituted or substituted with one or more substituents independently selected from the group consisting of a halogen atom, a C1-6 alkyl group, a C1-6 alkoxy group and a cyano group)] and is a group represented by}, or a salt thereof.

62. The X is a methyl group, and Q ５ to Q ８ is a hydrogen atom, the compound according to claim 61, or a salt thereof.

63. The R １ and R ２ are hydrogen atoms, the Y is NR ３ R ４ , and the R ３ and R ４ , together with the nitrogen atom to which they are attached, form morpholine, and Q ５ to Q ８ are hydrogen atoms, the compound according to claim 61 or 62, or a salt thereof.

64. The phosphorus-containing group is a cyanoethoxy(diisopropylamino)phosphino group or a hydroxyphosphinyl group, the compound according to any one of claims 61 to 63, or a salt thereof.

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