Modified oligonucleotides and double-stranded RNA

Abstract

The technology described herein relates to modified oligonucleotides and double-stranded RNAs, such as siRNAs, compositions and kits containing them, and methods of using them to inhibit target genes. TIFF2025534336000264.tif79159

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 412,000, filed September 30, 2022, and U.S. Provisional Application No. 63 / 451,486, filed March 10, 2023, the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in XML format, which is incorporated by reference in its entirety. This XML copy, created on September 29, 2023, is named "051058-000100WOPT_SL.xml" and is 4,106,746 bytes in size.

[0003] Technical Field The technology described herein relates to modified oligonucleotides and double-stranded RNAs, such as siRNAs, compositions and kits containing them, and methods of using them to inhibit target genes. [Background technology]

[0004] background There remains a need in the art for oligonucleotides and siRNAs with improved activity and / or pharmacodynamics. The present disclosure addresses some of these needs. Summary of the Invention

[0005] overview In one aspect, the present specification provides a double-stranded nucleic acid (for example, dsRNA) comprising antisense strand and sense strand, wherein antisense strand and sense strand are complementary to each other and form a double-stranded region, for example, a double-stranded region of at least 15 base pairs.Antisense strand comprises a ligand at its 3' end and comprises at least one nuclease-resistant modification at each end.In other words, antisense strand comprises a ligand at its 3' end, comprises at least one nuclease-resistant modification at its 3' end, and comprises at least one nuclease-resistant modification at its 5' end.

[0006] In some embodiments of any one of the aspects described herein, the sense strand also comprises at least one nuclease-resistant modification. For example, the sense strand comprises at least one nuclease-resistant modification at its 5'-end. In another non-limiting example, the sense strand comprises at least one nuclease-resistant modification at its 3'-end. In yet another non-limiting example, the sense strand comprises at least one nuclease-resistant modification at its 3'-end and at least one nuclease-resistant modification at its 5'-end.

[0007] As used herein, nuclease-resistant modification refers to a modification that increases the stability of nucleic acid (e.g., dsRNA) against degradation by nuclease (e.g., endonuclease or exonuclease).In other words, nuclease-resistant modification refers to a modification that inhibits or reduces the cleavage of nucleic acid by endonuclease or exonuclease compared to the cleavage of dsRNA that lacks that modification.Generally, nuclease-resistant modification is a modified internucleoside linkage, a modified sugar moiety, and / or a modified nucleobase.In some embodiments of any one of the aspects described herein, nuclease-resistant modification is a modified internucleoside linkage, for example, an internucleoside linkage other than phosphate ester.For example, nuclease-resistant modification is a phosphorothioate internucleoside linkage or a phosphorodithioate internucleoside linkage.

[0008] In some embodiments, the nuclease resistant modification is a 2'-5' linked nucleotide, e.g. TIFF2025534336000002.tif35128, where B is an optionally modified nucleobase and R is -OH or a sugar modification described herein (e.g., -F, -OMe).

[0009] In some embodiments, the nuclease resistant modification is an L-nucleotide, TIFF2025534336000003.tif37128, where B is an optionally modified nucleobase and R is -OH or a sugar modification described herein (e.g., -F, -OMe).

[0010] In another aspect, provided herein is a compound of formula (I): TIFF2025534336000004.tif32128 is provided.

[0011] In the compounds of formula (I), B is an optionally modified nucleobase.

[0012] In the compound of formula (I), X S is O, CH, S, or NH. In some embodiments of any one of the aspects described herein, X S is O or CH2. For example, X S is O.

[0013] In the compounds of formula (I), R 5 Ha-L 1 -R H or -ON(R 13 )R 14 where L 1 is a bond, -L 3 -, C 1~30 Alkylene, C 2~30 Alkenylene, C 2~30 Alkynylene, *-L 3 -C 1~30 Alkylene *-L 3 -C 2~30 Alkenylene, or *-L3 -C 2~30 alkynylene, and L 3 is -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(X L3 )(Y L3 R L3B )- and R L3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or nitrogen protecting group, X L2 is O or S, and Y L3 is O, S, NH, or a bond, and R L3B is H or optionally substituted alkyl, and * is R H is a bond to R H is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally the heterocyclyl contains at least one nitrogen atom or R H teeth TIFF2025534336000005.tif16128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylamino alkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars, and R 13 and R 14 is independently -L 2 -R H2 where L 2is the linker and R H2 is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally R 13 and R 14 At least one of them is -L 2 -R H2 is.

[0014] In some compounds of formula (I), R 5 Ha-L 1 -R H is.

[0015] In some compounds of formula (I), L 1 is L 3 For example, L 1 -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, or -P(X L3 )(Y L3 R L3B )-.

[0016] In some compounds of formula (I), L 1 is O or C 1~30 alkylene. For example, L 1 is O. In some other non-limiting examples, L 1 Ha-(CH2) n -, where n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30, e.g., n is 1, 2, 3, 4, 5, or 6). 1 is methylene, i.e., -CH2-.

[0017] In some compounds of formula (I), R His an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1 or 2 additional heteroatoms independently selected from N, O and S. In some compounds of formula (I), R H teeth TIFF2025534336000006.tif13128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0018] In some compounds of formula (I), R H teeth TIFF2025534336000007.tif13128, where X is O.

[0019] In some other compounds of formula (I), R H teeth TIFF2025534336000008.tif13128, where X is NR L In some further embodiments of these compounds, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. In still other embodiments of these compounds, R L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0020] In some compounds of formula (I), RH teeth TIFF2025534336000009.tif13128, where X is O.

[0021] In some other compounds of formula (I), R H teeth TIFF2025534336000010.tif13128, where X is NR L In some further embodiments of these compounds, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. In still other embodiments of these compounds, R L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0022] In some compounds of formula (I), R 5 HA-ON(R 13 )R 14 R 5 -ON(R 13 )R 14 If R 13 and R 14 It should be noted that R may be the same or different. Thus, in some compounds of formula (I), R 13 and R 14 are the same. In some other compounds of formula (I), R 13 and R 14 is different.

[0023] In some compounds of formula (I) described herein, R 13 and R 14 One or both of the -L 2 -R H2 It can be.

[0024] In some compounds of formula (I), L 2 is a bond or an optionally substituted alkylene. For example, L 2 is a bond. In some other compounds of formula (I), L 2 -Z-(CH2) m -, where Z is absent or is aryl, heteroaryl, cycloalkyl, or heterocyclyl, and m is 0 or an integer selected from 1 to 20 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., m is 1, 2, 3, 4, 5, or 6). For example, L 2 Ha-(CH2) m -or-(CH2) m -phenyl-.

[0025] In some compounds of formula (I), R 13 and R 14 At least one of (e.g., one or both) is -(CH2) m -R H2 or The file is TIFF2025534336000011.tif27128.

[0026] In some compounds of formula (I), R H2 is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S. For example, R H2 teeth TIFF2025534336000012.tif13128, where X is O, NR L , S, or CH2, and R Lis hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0027] In some compounds of formula (I), R H2 teeth TIFF2025534336000013.tif13128, where X is O.

[0028] In some other compounds of formula (I), R H2 teeth TIFF2025534336000014.tif13128, where X is NR L In some further embodiments, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0029] In some compounds of formula (I), R 13 and R 14 One of the groups is an optionally substituted C1-C6 alkyl. For example, R 13 and R 14 One of the groups is methyl.

[0030] In some embodiments of the compounds of Formula (I), R 13 and R 14One of them is -L 2 -R H2 and the other is an optionally substituted C1-C6 alkyl (for example, methyl).

[0031] In some compounds of formula (I), R 13 and R 14 One of the TIFF2025534336000015.tif22128, R 13 and R 14 the other is C1-C6 alkyl, TIFF2025534336000016.tif22128.

[0032] In some embodiments of any one of the aspects described herein, X P is -P(X)(OR V )2, where each X is independently O or S, and each R V is H or an oxygen protecting group. For example, R 5 -CH=CH-P(X)(OR V )2, where each X is independently O or S, and each R V are independently H or an oxygen protecting group.

[0033] Optionally, X is O. For example, R 5 -CH=CH-P(O)(OR V )2. In some embodiments, R 5 is -CH=CH-P(O)(OH). In some other embodiments, R 5 -CH=CH-P(O)(OR V )2, where each R V are independently oxygen protecting groups. For example, R 5 -CH=CH-P(O)(OR V )2, where each R V is independently 4-pentenyloxymethyl (POM). In yet some other embodiments, R 5 -CH=CH-P(O)(OH)(OR V ), where RV is an oxygen protecting group.

[0034] Optionally, X is S. For example, R 5 -CH=CH-P(S)(OR V )2. In some embodiments, R 5 is -CH=CH-P(S)(OH). In some other embodiments, R 5 -CH=CH-P(S)(OR V )2, where each R V are independently oxygen protecting groups. For example, R 5 -CH=CH-P(S)(OR V )2, where each R V is independently 4-pentenyloxymethyl (POM). In yet some other embodiments, R 5 -CH=CH-P(S)(OH)(OR V ), where R V is an oxygen protecting group.

[0035] In the compounds of formula (I), R 2 is hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9), a ligand, a linker covalently bonded to one or more ligands, a solid support, a linker, or a linker covalently bonded to a solid support. In some compounds of formula (I), R 2 is hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, dialkylamino, -OC 4~30 Alkyl-ON(CH2R8)(CH2R9), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), alkoxyoxycarboxylate. In some compounds of formula (I), R 2 is hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, reactive phosphorus(III) group, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, -OC 4~30 Alkyl-ON(CH2R8)(CH2R9), -OC 4~30 In some compounds of formula (I), R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, 2-methoxyethoxy, C 6~24 Alkyl (e.g., nC 6~24 alkyl), or a reactive phosphorus(III) group. In some compounds of formula (I), R 2is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, 2-methoxyethoxy, or a reactive phosphorus(III) group. In some compounds of formula (I), R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro or methoxy. In some compounds of formula (I), R 2 is hydrogen, fluoro or methoxy.

[0036] In the compounds of formula (I), R 3 is hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), a ligand, a linker covalently bonded to one or more ligands, a solid support, a linker, or a linker covalently bonded to a solid support. In some compounds of formula (I), R 3 is hydrogen, hydroxyl, protected hydroxyl, phosphate group, reactive phosphorus(III) group, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, dialkylamino, -OC4~30 Alkyl-ON(CH2R8)(CH2R9), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), alkoxyoxycarboxylate. For example, R 3 is a reactive phosphorus(III) group, hydrogen, hydroxyl, halogen, protected hydroxyl, phosphate group, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy or 2'-methoxyethoxy), alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, -OC 4~30 Alkyl-ON(CH2R8)(CH2R9), -OC 4~30 In some compounds of formula (I), R 3 is a reactive phosphorus(III) group, hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, 2-methoxyethoxy, or C 6~24 Alkyl (e.g., nC 6~24 In some compounds of formula (I), R 3 is a reactive phosphorus(III) group, hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy. In some compounds of formula (I), R 3 is a reactive phosphorus(III) group. In some compounds of formula (I), R 3 is a phosphoramidite group such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0037] In the compounds of formula (I), R 2 and R3 Note that at most one of the groups in R is a reactive phosphorus(III) group. 3 is the only reactive phosphorus(III) group.

[0038] In some compounds of formula (I), R 4 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 For example, R in formula (I) is alkoxy. 4 is H.

[0039] In some compounds of formula (I), R 4 and R 2 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v -2', and Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )- and R 10 and R 11 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, and R 12 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30alkyl-COH, or a nitrogen protecting group, and v is 1, 2, or 3. For example, R 2 and R 4 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v -2'.

[0040] In some compounds of formula (I), R 4 and R 3 C, together with the atoms to which they are attached, may be substituted 3~8 Cycloalkyl, optionally substituted C 3~8 It forms a cycloalkenyl or an optionally substituted 3- to 8-membered heterocyclyl.

[0041] In some embodiments of any one of the aspects described herein, the compound of Formula (I) has the formula (IA)-(ID): The compound is selected from TIFF2025534336000017.tif82142.

[0042] In some compounds of formula (I), (IA), (IB), (IC) or (ID), X S is O and R 2 and R 4 together form 4'-YC(R 10 R 11 ) v -2' and R 3 is a reactive phosphorus(III) group, a hydroxyl or a protected hydroxyl.

[0043] In some compounds of formula (I), (IA), (IB), (IC) or (ID), X S is O and R 2 are H, -OMe, -F, and R 3 is a reactive phosphorus(III) group, hydroxyl or protected hydroxyl, and R 4 is H.

[0044] In some embodiments of the various aspects described herein, the compound of Formula (I) is a compound of Formula (IE): TIFF2025534336000018.tif39128.

[0045] In some compounds of formula (IE), R 3 is a reactive phosphorus(III) group, a hydroxyl, or a protected hydroxyl, and R 5 Ha-L 1 -R H and X S , B, Y, R 10 and R 11 is as defined for formula (I).

[0046] In some embodiments of the various aspects described herein, the compound of formula (IE) is 1 ) or (IE 2 ) compounds: TIFF2025534336000019.tif47142, wherein n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R L is H, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0047] Expression (IE 1 ) or (IE 2 In some compounds ofs is O, Y is O, and R 10 and R 11 One of the groups is H and the other is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. For example, R 10 and R 11 One of is H and the other is H or linear, cyclic or branched alkyl (eg, methyl, propyl, isopropyl, etc.).

[0048] In some embodiments of the various aspects described herein, the compound of formula (IE) is a compound of formula (I-Ea), (I-Eb), or (I-Ec): TIFF2025534336000020.tif82138.

[0049] In some compounds of formula (IE), R 3 is a reactive phosphorus(III) group, a hydroxyl, or a protected hydroxyl, and X S , B, Y, R 10 and R 11 is as defined for formula (I).

[0050] In some embodiments of the various aspects described herein, the compound of formula (IE) is 3 ) or (IE 4 ) compounds: TIFF2025534336000021.tif42135, wherein n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R L is H, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0051] In some embodiments of the various aspects described herein, the compound of formula (I-Ea) is a compound of formula (I-Ed) or (I-Ee): The file is TIFF2025534336000022.tif43148.

[0052] In some embodiments of the various aspects described herein, the compound of formula (I-Eb) is a compound of formula (I-Ef): The file is TIFF2025534336000023.tif44128.

[0053] In some embodiments of the various aspects described herein, the compound of formula (I-Ec) is a compound of formula (I-Eg): The file is TIFF2025534336000024.tif37128.

[0054] Formula (I-Ed), (IE 3 ), (IE 4 In some compounds of (I-Ee), (I-Ef) and / or (I-Eg), R 3is a reactive phosphorus(III) group, a hydroxyl or a protected hydroxyl. For example, the formula (I-Ed), (IE 3 ), (IE 4 In some compounds of (I-Ee), (I-Ef) and / or (I-Eg), R 3 -OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0055] In some embodiments of the various aspects described herein, R 4 and R 2 together, 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v R unless -2' 5 is not morpholin-4-yl.

[0056] In some embodiments of the various aspects described herein, R 2 is H, hydroxyl, protected hydroxyl, alkoxy, or halogen, and R 3 is a hydroxyl, a protected hydroxyl, or a reactive phosphorus(III) group, and R 4 is H and X S If is O, then R 5 is not morpholin-4-yl.

[0057] Oligonucleotides The compound of formula (I) is useful in the synthesis of oligonucleotide.Therefore, in another aspect, the present invention provides the oligonucleotide prepared by using the compound of formula (I).For example, the oligonucleotide comprises the nucleoside of formula (II).Therefore, in another aspect, the present invention provides the nucleoside of formula (II): Oligonucleotides comprising at least one TIFF2025534336000025.tif32128 are provided.

[0058] In the nucleoside of formula (II), B is an optionally modified nucleobase.

[0059] In the nucleoside of formula (II), X S is O, CH, S, or NH. In some embodiments of any one of the aspects described herein, X S is O or CH2. For example, X S is O.

[0060] In the nucleoside of formula (II), R 5 Ha-L 1 -R H or -ON(R 13 )R 14 where L 1 is a bond, -L 3 -, C 1~30 Alkylene, C 2~30 Alkenylene, C 2~30 Alkynylene, *-L 3 -C 1~30 Alkylene *-L 3 -C 2~30 Alkenylene, or *-L 3 -C 2~30 alkynylene, and L 3 -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(X L3 )(Y L3 R L3B )- and R L3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or nitrogen protecting group, XL2 is O or S, and Y L3 is O, S, NH, or a bond, and R L3B is H or optionally substituted alkyl, and * is R H is a bond to R H is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally the heterocyclyl contains at least one nitrogen atom or R H teeth TIFF2025534336000026.tif16128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylamino alkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars, and R 13 and R 14 is independently -L 2 -R H2 where L 2 is the linker and R H2 is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally R 13 and R 14 At least one of them is -L 2 -R H2 is.

[0061] In some nucleosides of formula (II), R 5 Ha-L 1 -R H is.

[0062] In some nucleosides of formula (II), L 1 is L 3 For example, L 1 is -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, or -P(X L3 )(Y L3 R L3B )-.

[0063] In some nucleosides of formula (II), L 1 is O or an optionally substituted alkylene. For example, L 1 is O. In some other non-limiting examples, L 1 Ha-(CH2) n -, where n is 0 or an integer selected from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., n is 1, 2, 3, 4, 5, or 6). In some embodiments of any one of the aspects described herein, L 1 is methylene, i.e., -CH2-.

[0064] In some nucleosides of formula (II), R H is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S. For example, R H teeth TIFF2025534336000027.tif13128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0065] In some nucleosides of formula (II), R H teeth TIFF2025534336000028.tif13128, where X is O.

[0066] In some other nucleosides of formula (II), R H teeth TIFF2025534336000029.tif13128, where X is NR L In some further embodiments of these compounds, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. In still other embodiments of these compounds, R L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0067] In some nucleosides of formula (II), R H teeth TIFF2025534336000030.tif13128, where X is O.

[0068] In some other nucleosides of formula (II), R H teeth TIFF2025534336000031.tif13128, where X is NR L In some further embodiments of these compounds, R Lis H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. In still other embodiments of these compounds, R L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0069] In some nucleosides of formula (II), R 5 HA-ON(R 13 )R 14 R 5 -ON(R 13 )R 14 If R 13 and R 14 Note that R can be the same or different. Thus, in some nucleosides of formula (II), R 13 and R 14 are the same. In some other compounds of formula (I), R 13 and R 14 is different.

[0070] In some nucleosides of formula (II) described herein, R 13 and R 14 One or both of the -L 2 -R H2 It can be.

[0071] In some nucleosides of formula (II), L 2 is a bond or an optionally substituted alkylene. For example, L 2 is a bond. In some other compounds of formula (I), L 2 -Z-(CH2) m-, where Z is absent or is aryl, heteroaryl, cycloalkyl, or heterocyclyl, and m is 0 or an integer selected from 1 to 20 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., m is 1, 2, 3, 4, 5, or 6). For example, L 2 Ha-(CH2) m -or-(CH2) m -phenyl-.

[0072] In some nucleosides of formula (II), R 13 and R 14 At least one of (e.g., one or both) is -(CH2) m -R H2 or TIFF2025534336000032.tif27128.

[0073] In some compounds of formula (I), R H2 is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S. For example, R H2 teeth TIFF2025534336000033.tif13128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0074] In some nucleosides of formula (II), R H2 teeth TIFF2025534336000034.tif13128, where X is O.

[0075] In some other nucleosides of formula (II), R H2 teeth TIFF2025534336000035.tif13128, where X is NR L In some further embodiments, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0076] In some nucleosides of formula (II), R 13 and R 14 One of the groups is an optionally substituted C1-C6 alkyl. For example, R 13 and R 14 One of the groups is methyl.

[0077] In some embodiments of the nucleoside of Formula (II), R 13 and R 14 One of them is -L 2 -R H2 and the other is an optionally substituted C1-C6 alkyl (for example, methyl).

[0078] In some nucleosides of formula (II), R 13 and R 14 One of the TIFF2025534336000036.tif22128, R 13 and R 14 the other is C1-C6 alkyl, TIFF2025534336000037.tif22128.

[0079] In some nucleosides of formula (II), X P is -P(X)(OR V )2, where each X is independently O or S, and each R V is H or an oxygen protecting group. For example, in some nucleosides of formula (II), R 5 -CH=CH-P(X)(OR V )2, where each X is independently O or S, and each R V are independently H or an oxygen protecting group.

[0080] In some nucleosides of formula (II), X is O. For example, in some nucleosides of formula (II), R 5 -CH=CH-P(O)(OR V In some nucleosides of formula (II), R 5 is -CH=CH-P(O)(OH). In some other nucleosides of formula (II), R 5 -CH=CH-P(O)(OR V )2, where each R V are independently oxygen protecting groups. For example, in some nucleosides of formula (II), R 5 -CH=CH-P(O)(OR V )2, where each R V is independently 4-pentenyloxymethyl (POM). In some further nucleosides of formula (II), R 5 -CH=CH-P(O)(OH)(OR V ), where R V is an oxygen protecting group.

[0081] In some nucleosides of formula (II), X is S. For example, in some nucleosides of formula (II), R 5 -CH=CH-P(S)(OR VIn some nucleosides of formula (II), R 5 is -CH=CH-P(S)(OH). In some other embodiments, R 5 -CH=CH-P(S)(OR V )2, where each R V are independently oxygen protecting groups. For example, in some nucleosides of formula (II), R 5 -CH=CH-P(S)(OR V )2, where each R V is independently 4-pentenyloxymethyl (POM). In still other nucleosides of formula (II), R 5 -CH=CH-P(S)(OH)(OR V ), where R V is an oxygen protecting group.

[0082] In the nucleoside of formula (II), R 22 is a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 1~30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), a ligand, a linker covalently attached to one or more ligands, or a bond to an internucleotide linkage to a subsequent nucleoside, provided that R 22 and R 23In some embodiments, at least one, and only one, of R 22 is a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 1~30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino. For example, R 22 is hydroxyl, protected hydroxyl, halogen, or optionally substituted C 1~30 In some embodiments, R is alkoxy (e.g., methoxy, 2-methoxyethoxy). 22 is hydrogen, fluoro or methoxy.

[0083] In the nucleoside of formula (II), R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ), a phosphate group, a ligand, or a linker covalently attached to one or more ligands, provided that R 22 and R 23In some embodiments, at least one, and only one, of R 23 is the bond to the internucleotide linkage to the subsequent nucleotide.

[0084] In the nucleoside of formula (II), R 24 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 For example, R in the nucleoside of formula (II) can be alkoxy. 24 is H.

[0085] In some nucleosides of formula (II), R 24 and R 22 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v -2', and Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )- and R 10 and R 11 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, and R 12 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4Alkynyl, optionally substituted C 1~30 alkyl-COH, or a nitrogen protecting group, and v is 1, 2, or 3. For example, R 22 and R 24 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v It is -2.

[0086] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is selected from the group consisting of formulas (II-A) to (II-D): TIFF2025534336000038.tif88139.

[0087] In some nucleosides of formula (II), (II-A), (II-B), (II-C) or (II-D), X S is O and R 22 and R 24 together form 4'-YC(R 10 R 11 ) v -2' and R 23 is the bond to the internucleotide linkage to the subsequent nucleoside.

[0088] In some nucleosides of formula (II), (II-A), (II-B), (II-C) or (II-D), X S is O and R 22 is H, -OMe, or -F, and R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, and R 24 is H.

[0089] In the nucleosides of formula (II), (II-A), (II-B), (II-C) or (II-D), R 22 and R 23Note that at most one of the R is the bond to the internucleotide linkage to the subsequent nucleotide. For example, R 23 is the only bond to the internucleotide linkage to the subsequent nucleotide. In some other non-limiting examples, R 22 is the only bond to the internucleotide linkage to the subsequent nucleotide. 23 only the bond to the internucleotide linkage to the subsequent nucleotide.

[0090] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II) is a nucleoside of Formula (II-E): TIFF2025534336000039.tif39128.

[0091] In some nucleosides of formula (II-E), R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, and R 5 Ha-L 1 -R H and X S , B, Y, R 10 and R 11 is as defined for formula (II).

[0092] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-E) is 1 ) or (II-E 2 ) nucleosides: TIFF2025534336000040.tif45140, wherein: n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R Lis H, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0093] In some embodiments of any one of the aspects described herein, the compound of formula (II-E 1 ) or (II-E 2 ) nucleosides, X s is O, Y is O, and R 10 and R 11 One of the groups is H and the other is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. For example, R 10 and R 11 One of is H and the other is H or linear, cyclic or branched alkyl (eg, methyl, propyl, isopropyl, etc.).

[0094] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-E) is a nucleoside of Formula (II-Ea), (II-Eb), or (II-Ec): The file is TIFF2025534336000041.tif90153.

[0095] Some formulas (II-Ea), (II-E 1 ),(NO 2 ), (II-Eb) and / or (II-Ec) nucleosides, R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, and X S , B, Y, R10 and R 11 is as defined for formula (II).

[0096] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-Ea) is a nucleoside of Formula (II-Ed) or (II-Ee): TIFF2025534336000042.tif43142.

[0097] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-E) is 3 ) or (II-E 4 ) nucleosides: TIFF2025534336000043.tif45149, wherein n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R L is H, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0098] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-Eb) is a nucleoside of Formula (II-Ef): The file is TIFF2025534336000044.tif43128.

[0099] In some embodiments of any one of the aspects described herein, the nucleoside of Formula (II-Ec) is a nucleoside of Formula (II-Eg): The file is TIFF2025534336000045.tif37128.

[0100] In some embodiments of the various aspects described herein, R 24 and R 22 together, 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v R unless -2' 5 is not morpholin-4-yl.

[0101] In some embodiments of the various aspects described herein, R 22 is H, hydroxyl, protected hydroxyl, alkoxy, or halogen, and R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, and R 24 is H and X S If is O, then R 5 is not morpholin-4-yl.

[0102] In yet another aspect, provided herein is a double-stranded nucleic acid comprising a first strand and a second strand complementary to the first strand, wherein at least one of the first strand and the second strand is an oligonucleotide comprising a nucleoside of formula (II) described herein.

[0103] In some embodiments of the various aspects described herein, the double-stranded nucleic acid comprises a first strand and a second strand complementary to the first strand, wherein one of the first strand and the second strand is an oligonucleotide comprising a nucleoside of formula (II) described herein, and the other strand comprises a vinylphosphonate (VP) group (e.g., *=CH-X P , X P is a phosphate group and * is C5'), C 3~6Cycloalkylphosphonates (e.g., cyclopropylphosphonate), monophosphates ((HO)2(O)PO-5'), diphosphates ((HO)2(O)POP(HO)(O)-O-5'), triphosphates ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), monothiophosphates (phosphorothioates, (HO)2(S)PO-5'), monodithiophosphates (phosphorodithioates; (HO)(HS)(S)PO-5'), phosphorothiolates ((HO)2(O)PS-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), alkylphosphonates [(R P )(OH)(O)PO-5', R P is optionally substituted C 1~30 alkyl, for example, methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonates [(R P1 )(OH)(O)PO-5', R P1 is alkoxyalkyl, such as methoxymethyl (CH2OMe) or ethoxymethyl], (HO)2(X)PO[-(CH2) a -OP(X)(OH)-O] b -5' or (HO)2(X)PO[-(CH2) a -P(X)(OH)-O] b -5' or (HO)2(X)P-[-(CH2) a -OP(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl-terminated phosphates and phosphate mimics (e.g., HO[-(CH) a -OP(X)(OH)-O] b -5', H2N[-(CH2) a -OP(X)(OH)-O] b -5', H[-(CH2) a -OP(X)(OH)-O] b -5', Me2N[-(CH2) a -OP(X)(OH)-O]b -5', HO[-(CH2) a -P(X)(OH)-O] b -5', H2N[-(CH2) a -P(X)(OH)-O] b -5', H[-(CH2) a -P(X)(OH)-O] b -5', Me2N[-(CH2) a -P(X)(OH)-O] b -5') (wherein X is O or S, and a and b are each independently 1 to 10). For example, the double-stranded nucleic acid comprises a first strand and a second strand complementary to the first strand, one of which is an oligonucleotide comprising a nucleoside of formula (II) described herein, and the other strand comprises a vinylphosphonate group, e.g., an E-vinylphosphonate group, at its 5'-terminus.

[0104] In some embodiments of any one of the aspects described herein, the nuclease resistant modification is a nucleoside of Formula (II).

[0105] In some embodiments of any one of the aspects described herein, the oligonucleotide described herein comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, phosphorothioate internucleoside linkages. For example, the oligonucleotide comprises at least four phosphorothioate internucleoside linkages, e.g., at least six phosphorothioate internucleoside linkages, or at least eight phosphorothioate internucleoside linkages.

[0106] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate internucleoside linkages. For example, the dsRNA comprises at least four phosphorothioate internucleoside linkages, for example, at least six phosphorothioate internucleoside linkages, or at least eight phosphorothioate internucleoside linkages.

[0107] It should be noted that phosphorothioate internucleoside linkages can be present in one or both strands. Furthermore, phosphorothioate internucleoside linkages can be present anywhere in the chain. For example, phosphorothioate internucleoside linkages can be present at one end of the chain, at both ends of the chain, at both ends of the chain and at an internal position, or at both ends of the chain and at an internal position. Preferably, phosphorothioate internucleoside linkages are present at both ends of the chain.

[0108] In some embodiments, the antisense strand comprises at least one, e.g., two, three, or four or more, phosphorothioate internucleoside linkage. For example, the antisense strand comprises four or more phosphorothioate internucleoside linkages. In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second positions, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second positions, counting from the 5' end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second positions and between the second and third positions, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second positions, counting from the 5' end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second and second and third positions, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second and second and third positions, counting from the 5' end of the strand. In some further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second, second and third, and third and fourth positions, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second positions, counting from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second positions, counting from the 3' end of the strand, and between the first and second and second and third positions, counting from the 5' end of the strand. In other further embodiments of any one of the aspects described herein, the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second positions, counting from the 3' end of the strand, and between the first and second, second and third, and third and fourth positions, counting from the 5' end of the strand.

[0109] Like the antisense strand, the sense strand can also include one or more, e.g., two, three, or four or more, phosphorothioate internucleoside linkages. For example, the sense strand includes a phosphorothioate internucleoside linkage between the first and second positions, counting from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the sense strand includes a phosphorothioate internucleoside linkage between the first and second positions, counting from the 5' end of the strand, and between the first and second positions, counting from the 3' end of the strand.

[0110] In some further embodiments of any one of the aspects described herein, the sense strand comprises phosphorothioate internucleoside linkages between the first and second and second and third positions, counting from the 5' end of the strand. For example, the sense strand comprises phosphorothioate internucleoside linkages between the first and second and second and third positions, counting from the 5' end of the strand, and between the first and second and second and third positions, counting from the 3' end of the strand.

[0111] In some embodiments of any one of the aspects described herein, the antisense strand and the sense strand can independently be at least about 18, e.g., about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides in length, or longer. For example, the antisense strand is about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides in length. In some embodiments of any one of the aspects described herein, the antisense strand is about 22, about 23, or about 25 nucleotides in length.

[0112] Similar to the antisense strand, in some embodiments of any one of the aspects described herein, the sense strand is at least about 16, for example, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides in length, or longer. For example, the sense strand is about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is about 21 nucleotides in length.

[0113] In some embodiments of any one of the aspects described herein, the antisense strand is 22, 23, or 25 nucleotides in length, and the sense strand is 21 nucleotides in length.

[0114] In some embodiments of any one of the aspects described herein, the sense strand is 15 nucleotides in length and the antisense strand is 18, 19, 20, 21, or 22 (e.g., 20) nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 19 nucleotides in length and the antisense strand is 19, 20, or 21 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20 nucleotides in length and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 21 nucleotides in length and the antisense strand is 21, 22, or 23 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20-24 (e.g., 22) nucleotides in length and the antisense strand is 34-38 (e.g., 36) nucleotides in length.

[0115] In some embodiments of the various aspects described herein, the double-stranded region of the dsRNA can be at least about 18, e.g., about 19, about 20, about 21, about 22, about 23, about 24, or about 25 base pairs, or more, e.g., about 21 base pairs.

[0116] In some embodiments of any one of the aspects described herein, the antisense strand is about 21, about 22, about 23, about 24, or about 25 nucleotides in length, the sense strand is about 21 nucleotides in length, and the dsRNA comprises a double-stranded region of at least 18, e.g., 19, 20, or 21 base pairs, e.g., 21 base pairs.

[0117] Generally, the ligand is linked to the 3'-end of the antisense strand. The ligand can be linked to any available position of the nucleotide at the 3'-end, i.e., the first nucleotide (counting from the 3'-end) of the antisense strand. For example, the ligand can be attached to the 3'-hydroxyl, 2'-hydroxyl (if present), or a position in the nucleobase. In some embodiments of any one of the aspects described herein, the ligand is linked to the 3'-hydroxyl of the first nucleotide counting from the 3'-end of the antisense strand. The ligand can be linked to the 3'-end of the antisense strand directly, i.e., via a bond, or via a linker.

[0118] It should be noted that the ligand or the linker attached to the ligand can be linked to the 3'-end of the antisense strand via any modified or unmodified internucleoside linkage known and available in the art. For example, the ligand or the linker attached to the ligand can be linked to the 3'-end of the antisense strand via any negatively charged moiety. For example, the ligand or the linker attached to the ligand can be linked to the 3'-end of the antisense strand via a phosphodiester (PO), phosphorothioate (PS), phosphorodithioate (PS2), PN (e.g., RSO2-N=P(OH) type, or (HO)P-NHR or (HO)P-NR2, where R is an aliphatic (e.g., C1-20 alkyl), cycloaliphatic, heterocyclic, aromatic, or heteroaromatic group, respectively, but is not limited to these. or where both R groups, together with the nitrogen to which they are attached, form a 4- to 10-membered monocyclic or bicyclic heterocyclic group, which may have one or two additional heteroatoms selected from O, N, and S, and which may be substituted. Optional substituents include halogen, cyano, nitro, azido, hydroxy, amino, carboxy, oxo (=O), thia (=S), imino (=N(H)), C 1~6 Alkylimino (=N(R)), C 1~6 Alkylamino (R(H)N-), diC 1~6 Alkylamino (RN-), C 1~6 Alkyl, C 1~6 Alkoxy, C 1~6 Acyl (RC(O)-), C 1~6 Alkyl ester (ROC(O)-), amide (H2NC(O)-), C 1~6 Alkylamide (R(H)NC(O)-), diC 1~6 Alkylamide (RNC(O)-), C 1~6 and acylamino (RC(O)N(H)—) linkages.

[0119] In some embodiments of any one of the aspects described herein, the ligand or the linker attached to the ligand is linked to the 3'-end of the antisense strand via a phosphorothioate internucleoside linkage.

[0120] The linker can be selected to position the ligand away from the PAZ domain of Ago. Thus, in some embodiments of any one of the aspects described herein, the linker connecting the ligand to the 3'-end of the antisense strand is about 5 angstroms to about 250 angstroms in length. For example, the linker connecting the ligand to the 3'-end of the antisense strand is about 10 angstroms to about 200 angstroms in length, e.g., about 15 angstroms to about 150 angstroms, about 20 angstroms to about 100 angstroms, about 25 angstroms to about 75 angstroms, about 5 angstroms to about 50 angstroms, about 10 angstroms to about 40 angstroms, or about 20 angstroms to about 30 angstroms in length.

[0121] In some embodiments of any one of the aspects described herein, the linker has a chain length of at least 6 atoms. For example, the linker has a chain length of at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 atoms or more. In some embodiments of any one of the aspects described herein, the linker has a chain length of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms.

[0122] In some embodiments of any one of the aspects described herein, the ligand is linked to the 3' end of the antisense strand via a linker, e.g., the ligand is linked to the 3' end of the antisense strand via a hydrophobic linker.

[0123] In various aspects described herein, the linker can include a carrier tethered to the carrier. In some embodiments, the carrier includes a hydrogen bond acceptor (e.g., a tertiary amide or a tertiary amine). In some embodiments, the carrier includes a pyrrolidine ring.

[0124] The present inventors have discovered that, inter alia, the pharmacokinetic (PK) / pharmacodynamic (PD) properties of dsRNAs comprising a ligand linked to the 3'-end of the antisense strand can be improved by including a second ligand in the dsRNA. Thus, in some embodiments of any one of the aspects described herein, the dsRNA comprises a second ligand. The second ligand can be attached or linked to the sense strand or the antisense strand. Preferably, the second ligand is linked to the sense strand. In some embodiments of any one of the aspects described herein, the second ligand is linked to the 3'-end of the sense strand. In other embodiments of any one of the aspects described herein, the second ligand is linked to the 5'-end of the sense strand. Note that the ligand linked to the antisense strand and the second ligand can be the same or different. Preferably, the ligand linked to the antisense strand and the second ligand are different.

[0125] Various aspects of the present invention include ligands such as targeting ligands, PK regulators, or endosomolytic ligands.Therefore, the ligand linked to the 3' end of antisense strand can be a targeting ligand, PK regulators, or endosomolytic ligands.Preferably, the ligand linked to the 3' end of antisense strand is a targeting ligand, such as monovalent or multivalent N-acetylgalactosamine (GalNac).

[0126] When there is a second ligand, it can be a targeting ligand, a PK regulator or an endosomolytic ligand.For example, the second ligand is a ligand that can bind to serum protein, such as serum albumin.The exemplary ligand that can bind to serum albumin includes, but is not limited to, iodipamide, azapropazone, indomethacin, tiblone (TIB), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), DIS, oxyphenbutazone, phenylbutazone, warfarin, indoxyl sulfate, diflunisal, halothane, ibuprofen, and diazepam, propofol.

[0127] In some embodiments of any one of the aspects described herein, the ligand linked to the sense strand, i.e., the second ligand, is a PK modulator.

[0128] In some embodiments of any one of the aspects described herein, the ligand linked to the sense strand, i.e., the second ligand, is a mannose receptor ligand (e.g., multivalent mannose).

[0129] In some embodiments of any one of the aspects described herein, the ligand linked to the sense strand, i.e., the second ligand, is a folate ligand.

[0130] In some embodiments of any one of the aspects described herein, the ligand linked to the 3' end of the antisense strand is a targeting ligand, e.g., monovalent or multivalent GalNAc, and the second ligand is a PK modulator, e.g., ibuprofen.

[0131] In some embodiments of any one of the aspects described herein, the ligand linked to the 3' end of the antisense strand is a targeting ligand, e.g., monovalent or multivalent GalNAc, and the ligand linked to the sense strand is a mannose receptor ligand (e.g., mannose).

[0132] In some embodiments of any one of the aspects described herein, the ligand linked to the 3' end of the antisense strand is a targeting ligand, e.g., monovalent or multivalent GalNAc, and the ligand linked to the sense strand is a folate ligand.

[0133] In any one of the aspects described herein, each ligand can be independently selected from the group consisting of peptides, centrins, antibodies (e.g., anti-CD4 antibodies and anti-CD117 antibodies), antibody fragments, T cell targeting ligands, B cell targeting ligands, cancer cell targeting ligands (e.g., DUPA, folate, and RGD), spleen targeting functionality, lung targeting functionality, bone marrow targeting functionality, phage-displayed peptides, cell-penetrating peptides (CPPs), integrin ligands, polyanionic ligands, polycationic ligands, monovalent and polyvalent carbohydrates (e.g., GalNAc, mannose, mannose-6 phosphate, mucose, and mulucose), kidney targeting ligands, BBB-penetrating ligands, lipids, and amino acids (e.g., L-amino acids, D-amino acids, and β-amino acids).

[0134] It should be noted that the double-stranded RNA described can comprise one or more additional nucleic acid modifications, such as nucleic acid base modification, sugar modification, inter-sugar linkage modification, or any combination thereof.Therefore, in some embodiments of any one of the aspects described herein, dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more 2'-fluoro nucleotides.For example, antisense strand and / or sense strand independently comprise at least one, for example, 2, 3, 4 or 5 or more 2'-fluoro nucleotides.

[0135] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand. For example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand. In another non-limiting example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand. In some further examples, 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.

[0136] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0137] In some embodiments of any one of the aspects described herein, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at positions 11, 13, and 15, counting from the 3' end of the sense strand. For example, the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, 13, and 15, counting from the 3' end of the sense strand.

[0138] In some embodiments of any one of the aspects described herein, the sense strand comprises 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, and 13, counting from the 3' end of the sense strand.

[0139] In some embodiments of any one of the aspects described herein, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at positions 11, 13, and 15, counting from the 3' end of the sense strand. For example, the antisense strand comprises 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand, and the sense strand comprises 2'-fluoro nucleotides at positions 7, 9, and 11, counting from the 5' end of the sense strand, or at positions 11, 13, and 15, counting from the 3' end of the sense strand. In another example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 9, 14, and 16 positions counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least 7, 9, and 11 positions counting from the 5' end of the sense strand, or at least 11, 13, and 15 positions counting from the 3' end of the sense strand. In yet another example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 8, 9, 14, and 16 positions counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least 7, 9, and 11 positions counting from the 5' end of the sense strand, or at least 11, 13, and 15 positions counting from the 3' end of the sense strand.

[0140] In some further non-limiting examples, the antisense strand contains 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at positions 7, 9, and 11 from the 5' end of the sense strand or at positions 11, 12, 13, and 15 from the 3' end of the sense strand. For example, the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5' end of the sense strand or at positions 11, 12, 13, and 15 from the 3' end of the sense strand. In another example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 9, 14, and 16 positions counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least 7, 9, 10, and 11 positions counting from the 5' end of the sense strand or at least 11, 12, 13, and 15 positions counting from the 3' end of the sense strand. In yet another example, the antisense strand contains 2'-fluoro nucleotides at least 2, 6, 8, 9, 14, and 16 positions counting from the 5' end of the antisense strand, and the sense strand contains 2'-fluoro nucleotides at least 7, 9, 10, and 11 positions counting from the 5' end of the sense strand or at least 11, 12, 13, and 15 positions counting from the 3' end of the sense strand.

[0141] The dsRNAs described herein can contain one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-deoxy (i.e., 2'-H or DNA) nucleotides. For example, the antisense strand and / or the sense strand independently contain at least one, for example, 2, 3, 4, or 5 or more 2'-deoxy (i.e., 2'-H or DNA) nucleotide.

[0142] In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand.

[0143] In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, and a 2'-fluoro nucleotide at position 14 of the antisense strand.

[0144] dsRNA described herein can comprise one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more 2'-OMe nucleotides.For example, antisense strand and / or sense strand independently comprise at least one, for example, 2, 3, 4 or 5 or more 2'-OMe nucleotides.In some embodiments of any one of the aspects described herein, the remaining nucleotides in antisense strand, i.e., all except for the modifications specified herein, are 2'-OMe nucleotides.Similarly, in some embodiments of any one of the aspects described herein, the remaining nucleotides in antisense strand, i.e., all except for the modifications specified herein, are 2'-OMe nucleotides.

[0145] In some embodiments of any one of the aspects described herein, the antisense strand comprises a phosphate group or a phosphate analog or derivative thereof at its 5'-end. For example, the antisense strand comprises a 5'-vinylphosphonate nucleotide at its 5'-end. For example, the antisense strand comprises a 5'-E-vinylphosphate nucleotide at its 5'-end.

[0146] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, locked nucleic acid (LNA) or bridged nucleic acid (BNA) nucleotides. For example, the antisense strand and / or the sense strand independently comprise at least one, e.g., 2, 3, 4, or 5 or more, LNA or BNA nucleotides.

[0147] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, cyclohexene nucleic acid (CeNA) nucleotides. For example, the antisense strand and / or the sense strand independently comprise at least one, e.g., 2, 3, 4, or 5 or more, CeNA nucleotides.

[0148] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more, thermostabilizing modification. For example, the antisense strand and / or the sense strand independently comprise at least one, e.g., 2, 3, 4, or 5 or more, thermostabilizing modification.

[0149] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more abasic nucleotides. For example, the antisense strand and / or the sense strand independently comprise at least one, e.g., 2, 3, 4, or 5 or more abasic nucleotides.

[0150] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-deoxynucleotides. For example, the antisense strand and / or the sense strand independently comprise at least one, for example, 2, 3, 4, or 5 or more 2'-deoxynucleotides. In some embodiments, the antisense strand comprises one or more, for example, one, two, or more 2'-deoxynucleotides in the single-stranded overhang.

[0151] In some embodiments of any one of the aspects described herein, the dsRNA comprises at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more acyclic (e.g., unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or (S)-glycol nucleic acid (S-GNA)) nucleotide. For example, the antisense strand and / or the sense strand independently comprise at least one, e.g., 2, 3, 4, or 5 or more UNA and / or GNA nucleotides.

[0152] In some embodiments of any one of the aspects described herein, dsRNA comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more thermo-destabilizing modifications.For example, antisense strand and / or sense strand independently comprise at least one, for example, 2, 3, 4, or 5 or more thermo-destabilizing modifications.Exemplary thermo-destabilizing modifications include, but are not limited to, abasic nucleotides, 2'-deoxynucleotides, acyclic nucleotides (for example, UNA, GNA and (S)-GNA), 2'-5' linked nucleotides (3'-RNA), threose nucleotides (TNA), 2' gem Me / F nucleotides, and mismatches with the opposite nucleotide in the other strand.

[0153] In some embodiments of any one of the aspects described herein, the antisense strand comprises at least one thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2-9 from the 5' end). For example, the antisense strand comprises a thermodestabilizing modification at at least one of positions 6, 7, or 8 from the 5' end of the strand. In some embodiments of any one of the aspects described herein, the antisense strand comprises a thermodestabilizing modification at position 7 from the 5' end of the strand.

[0154] The double-stranded nucleic acid may have blunt ends and / or single-stranded overhangs at its ends. For example, the double-stranded nucleic acid may have a blunt end at the 5'-end of the antisense strand. In another example, the double-stranded nucleic acid may have a single-stranded overhang of 1 to 5 nucleotides at the 3'-end of the antisense strand, for example, the 3'-end of the antisense strand extends beyond the 5'-end of the sense strand.

[0155] In another aspect, provided herein are pharmaceutical compositions comprising the oligonucleotide or dsRNA molecules described herein, alone or in combination with a pharmaceutically acceptable carrier or excipient.

[0156] In yet another aspect, provided herein is a cell comprising an oligonucleotide or dsRNA molecule described herein.

[0157] In yet another aspect, provided herein is a gene silencing kit comprising an oligonucleotide or dsRNA molecule described herein.

[0158] Also provided herein is a method for silencing target gene in cell.This method includes: (i) introducing into cell the dsRNA molecule described herein, wherein one of the strands of dsRNA, for example antisense, comprises the nucleotide sequence that is substantially complementary to the nucleotide sequence of target gene; and / or (ii) the oligonucleotide described herein, comprises the nucleotide sequence that is substantially complementary to the nucleotide sequence of target gene.

[0159] In another aspect, the present specification provides a method for inhibiting or reducing the expression of a target gene in a subject.The method comprises: (i) administering to a subject the dsRNA molecule described herein, wherein one of the strands of the dsRNA, for example, antisense, comprises the nucleotide sequence that is substantially complementary to the nucleotide sequence of the target gene; and / or (ii) administering to a subject the oligonucleotide described herein, wherein the nucleotide sequence that is substantially complementary to the nucleotide sequence of the target gene. [Brief explanation of the drawings]

[0160] [Figure 1] Schematic diagram of siRNA in which GalNAc is conjugated to the 3' end of the sense strand (Figure 1A) and the 3' end of the antisense strand (Figure 1B). Deoxythymine residues are shown in blue, 2'-fluoro is shown in green, and 2'-O-methyl is shown in black. Phosphorothioate linkages are shown as orange lines. Figure 1C shows the chemical structure of the GalNAc ligand. [Figure 2] 1 shows the ASGPR binding affinity of GalNAc moieties conjugated to siRNA. [Figure 3A] Figures 3A-3C are graphs showing the in vitro activity of siRNAs targeting mTTR (Figure 3A), C5 (Figure 3B), and FXII (Figure 3C) by transfection (top row) or free uptake (bottom row). [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 4] 1 is a bar graph showing that siRNA with GalNAc conjugated to the 3' end of the antisense strand is effective in vivo. Percentage of circulating TTR protein after treatment of mice with GalNAc conjugate I or II compared to PBS-treated control mice (n=3). Blood samples were collected 4 days (purple) and 7 days (green) after dosing, and TTR protein levels were quantified by ELISA. Serum TTR protein levels from individual animals were normalized to the PBS-treated control group and are presented as the mean ± standard deviation. [Figure 5] Figure 1 shows relative circulating TTR protein after treatment with GalNAc conjugates II and VII compared to pre-dose levels. siRNA is shown schematically above the graph. The location of the phosphorothioate linkage is indicated by an orange line. C57BL / 6 mice were treated subcutaneously with 1 mg / kg II (blue) and 0.5 (green), 1 (red), and 2.5 (brown) mg / kg VII (n=3). Blood samples were collected 4, 7, 10, 14, and 21 days after dosing, and TTR was quantified by ELISA. Serum TTR protein levels from individual animals were normalized to pre-dose levels and are presented as the mean ± standard deviation. [Figure 6]Graphs (n=3) show the percentage of circulating TTR protein after treatment with GalNAc conjugates I (blue), II (orange), and VIII (purple) compared to pre-dose levels in C57BL / 6 mice. siRNAs are shown schematically on the right. Mice were treated with (Figure 6A) 2.5 mg / kg and (Figure 6B) 1 mg / kg subcutaneously. Blood samples were collected 7, 14, 21, and 28 days after dosing, and TTR protein was quantified by ELISA. Serum TTR protein levels from individual animals were normalized to pre-dose levels and are presented as the mean ± standard deviation. [Figure 7] Graphs showing the percentage of circulating TTR protein after treatment with GalNAc conjugates II (blue), IX (orange), and X (purple) compared to pre-dose levels in C57BL / 6 mice (n=3). Mice were treated subcutaneously with (Figure 7A) 2.5 mg / kg or (Figure 7B) 1.0 mg / kg. Blood samples were collected 7, 14, 21, and 28 days after dosing, and TTR protein was quantified by ELISA. Serum TTR protein levels from individual animals were normalized to pre-dose levels and are presented as the mean ± standard error. [Figure 8]Figure 8A shows liver siRNA levels in wild-type C57BL / 6 mice after a single subcutaneous administration of 1 mg / kg siRNA. Livers were collected 5 days after dosing, and siRNA levels were quantified by RT-qPCR. Sense strand (blue) and antisense strand (red) levels were assessed. Data are presented as the mean + / - standard deviation. Figure 8B shows the levels of siRNA loaded into Ago2 in wild-type C57BL / 6 mice after a single subcutaneous administration of 1 mg / kg siRNA. Livers were collected 5 days after dosing, and siRNA levels in Ago2 were quantified by RT-qPCR. Sense strand (blue) and antisense strand (red) levels were assessed. Data are presented as the mean + / - standard deviation. Figure 8C shows a comparison of mTTR mRNA knockdown in liver after a single SC administration of siRNA I, VIII, II, and VII in wild-type C57BL / 6 mice. Results are presented as the % mTTR mRNA remaining in liver after a single SC administration of 1 mg / kg. [Figure 9] Figure 9A shows that duplex (XIV) exhibits better potency and longer duration of action after 42 days compared to (II), (XIII), (XI), and (XII); (Figure 9A) 2.5 mg / kg (Figure 9B) 1.0 mg / kg. Chemistry used: 3'-GalNAc in the antisense strand, 8 PS for II and XI-XIV. [Figure 10] Figure 1 is a bar graph showing relative circulating TTR protein after treatment with GalNAc conjugates XV and XVI compared to pre-dose levels. siRNAs are shown schematically above the graph. The location of phosphorothioate linkages is indicated by orange lines. C57BL / 6 mice were treated subcutaneously with 1 mg / kg XV (blue) and 0.5 (green), 1 (red), and 2.5 (brown) mg / kg XVI (n=3). Blood samples were collected 4, 7, 10, 14, and 21 days after dosing, and TTR was quantified by ELISA. Serum TTR protein levels from individual animals were normalized to pre-dose levels and are presented as the mean ± standard deviation. [Figure 11]Figure 11A shows in vivo gene silencing of 3'-AS GalNAc (6PS and 8PS) conjugates of C5 siRNA: PD observed on day 5. Comparison of gene silencing of conjugates III, IV, and XVII in wild-type C57BL / 6 mice (n=3). Results are expressed as the % C5 protein remaining in the circulation after a single SC administration at a 1 mg / kg dose compared to pre-dose. Blood samples were collected 5 days after dosing for C5 protein assessment by ELISA. Serum C5 protein levels from individual animals were normalized to pre-dose levels and are presented as the mean ± standard error. Chemistry used in Figures 11A-C: III, parental, 3'-GalNAc in the sense strand (parental control), 6PS; IV, 3'-GalNAc in the antisense strand, 8PS; and XVII, 3'-GalNAc in the antisense strand, 6PS. Figure 11B shows tissue levels of siRNA-GalNAc conjugates in the liver at 24 hours after a single 1 mg / kg subcutaneous dose (SC, SD) in C57BL / 6 mice (n=3). siRNA levels were determined using a PCR-based assay. Figure 11C shows in vivo Ago2 loading of siRNA. [Figure 12] This figure shows the docking of a GalNAc linker attached to the 3' end of the siRNA guide strand, which interacts with the PAZ domain. The linker is positioned away from the PAZ domain. This explains how GalNAc is accommodated in the PAZ domain and how the hydroxyproline is stabilized within the PAZ domain. [Figure 13] The results of in vitro experiments (FIG. 13A) and in vivo studies (FIGS. 13B and 13C) are shown. Both single-stranded siRNAs XIX and XX were administered to wild-type C57BL / 6 mice to observe the dose response of mouse transthyretin mRNA (mTTR) by single subcutaneous administration of 3.0 mg / kg (FIG. 13B) and 10.0 mg / kg (FIG. 13C). Circulating mTTR protein levels were analyzed 3, 7, and 14 days after administration. [Figure 14] FIG. 1 is a schematic diagram of an exemplary chemically modified siRNA design. [Figure 15] 1 shows some exemplary chemically modified siRNAs in which GalNAc is conjugated to the 3'-end of the antisense and sense strands. [Figure 16] 16A and 16B are graphs showing that this evaluation architecture reveals (XIV) as the best construct at 2.5 mg / kg (FIG. 16A) and 1.0 mg / kg (FIG. 16B). [Figure 17] Figure 17 shows the activity of single-stranded siRNAs. Both single-stranded siRNAs XIX and XX were administered to wild-type C57BL / 6 mice by single subcutaneous administration of 3.0 mg / kg (Figure 17A) and 10.0 mg / kg (Figure 17B) to observe the dose response of mouse transthyretin mRNA (mTTR). Circulating mTTR protein levels were analyzed 3, 7, and 14 days after administration. [Figure 18] 1 depicts the experimental design to evaluate the effect of the ibuprofen ligand on the potency and PK of the conjugate. [Figure 19]

[0033] Figure 19 shows the in vivo activity of GalNAc-ibuprofen conjugates in wild-type mice. SEQ ID NOs are shown in Table 19. [Figure 20] Figure 19 depicts the binding of ibuprofen-conjugated siRNA to human serum albumin (HAS). SEQ ID NOs are shown in Table 19. [Figure 21] PK / PD analysis of GalNAc-ibuprofen conjugates bearing GalNAc and a hydrophobic PK enhancer (ibuprofen, which binds to albumin) is shown. [Figure 22-1] Various oligonucleotide sequences are represented. [Figure 22-2] See description of Figure 22-1. [Figure 23] Figure 1 shows in vivo TTR protein levels in serum samples over a 42 day period following subcutaneous (sc) administration of siRNAs with various ligands at the 5' and 3' ends. [Figure 24]1 shows in vivo TTR protein levels in serum samples over a 42-day period following intravenous (iv) administration of siRNAs with various ligands at the 5' and 3' ends. [Figure 25A] Figures 25A-25E depict exemplary ligands. Figure 25A shows various examples of ligands and representative L groups. Figure 25B shows several exemplary ligand aldehydes. Figure 25C shows several exemplary ligand acids. Figure 25D shows several exemplary multivalent mannose-based ligands, including acids for trivalent and hexavalent mannose, aldehydes for trivalent and hexavalent mannose, acids for hexavalent and multivalent mannose, and aldehydes for hexavalent and multivalent mannose. Figure 25E shows multivalent mannose-based ligands, including acids for hexavalent and multivalent mannose, aldehydes for hexavalent and multivalent mannose, acids for multivalent mannose, and aldehydes for multivalent mannose. [Figure 25B] See legend to Figure 25A. [Figure 25C] See legend to Figure 25A. [Figure 25D] See legend to Figure 25A. [Figure 25E] See legend to Figure 25A. [Figure 26] Various sense (S) and antisense (AS) strands with different ligands at the 5' and 3' ends are shown. [Figure 27] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 28] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 29] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 30] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 31] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 32] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 33] 1A-1D depict some exemplary aspects of the present disclosure. [Figure 34]1 is a bar graph showing that modification of the sense strand with a phosphorylation blocker enhances silencing in mice. Mice (n=3 per group) were treated with a single dose (3 mg kg-1) of siRNA I, III, IV, or V (Table 10) targeting Apob. Circulating Apob protein levels were quantified at 3, 7, 14, and 21 days. Levels were normalized to Gapdh. Data are expressed as a percentage of Apob in PBS-treated control animals. [Figure 35] 1 is a bar graph showing that antisense strand modification by Mo1 or Mo2 inhibits silencing. Mice (n=3 per group) were treated with a single dose (3 mg kg-1) of siRNA II, VI, VII, and VIII targeting Apob (Table 10). Circulating Apob protein levels were quantified at 3, 7, 14, and 21 days. Apob levels were normalized to Gapdh. Data are expressed as a percentage of Apob in PBS-treated control animals. [Figure 36] Figure 1 is a line graph showing that gene silencing activity is inhibited by Mo2 modification of the antisense strand. Percent luciferase expression in a TTR receptor assay as a function of siRNA concentration. The antisense strand of siRNA targeting TTR was modified with the indicated morpholino analogs. The parent strand had no 5' modification. [Figure 37] Figure 1 is a bar graph showing that extended morpholino modifications at the 5' position inhibit RISC loading. Total antisense RNA bound to recombinant human Ago2 as quantified by stem-loop RT-PCR. [Figure 38A]Figures 38A-38H are schematic diagrams showing that morpholino analogs disrupt the interaction between the 5' phosphate and the MID domain of Ago2. Figures 12A-12D show models of Ago2 bound to chains bearing (Figure 38A) Mo1, (Figure 38B) Mo2, (Figure 38C) Pip, and (Figure 38D) Mo3. Figure 38E shows an overlay of the complexes shown in Figures 138A-38D. Figure 38F shows potential hydrogen bond formation with Mo2. Figure 38G shows the Ago2 surface colored according to Coulomb potential (blue - positive, white - neutral). Figure 38H shows the Ago2 surface colored according to hydrophobicity (green - lowest and pink - highest). [Figure 38B] See legend to Figure 38A. [Figure 38C] See legend to Figure 38A. [Figure 38D] See legend to Figure 38A. [Figure 38E] See legend to Figure 38A. [Figure 38F] See legend to Figure 38A. [Figure 38G] See legend to Figure 38A. [Figure 38H] See legend to Figure 38A. [Figure 39] 1 is a bar graph showing a selection of 5'-morpholino-modified sense, antisense, and control strands in vivo. Mice (n=3 per group) were treated with a single dose (3 mg kg-1) of parent siRNA and duplexes I-VIII (Table 10) targeting Apob. Circulating Apob protein levels were quantified at 3, 7, 14, and 21 days. Apob levels were normalized to Gapdh mRNA. Data are expressed as percent of Apob in PBS-treated control animals. [Figure 40-1] FIG. 1 is a schematic diagram of a duplex targeting mTTR. [Figure 40-2] See description of Figure 40-1. [Figure 41-1] FIG. 1 is a schematic diagram of a duplex targeting F9. [Figure 41-2] See description of Figure 41-1. [Figure 42-1]FIG. 1 is a schematic diagram of an ApoB-targeting duplex. [Figure 42-2] See description of Figure 42-1. [Figure 43] Schematic diagram of control LNA duplexes against mTTR. SEQ ID NOs are shown in Table 19. [Figure 44A] Figures 44A-G show IC50 curves in PMH following transfection of mTTR-targeting duplexes: AD-57727 (Figure 44A), AD-68895 (Figure 44B), AD-617745 (Figure 44C), AD-617746 (Figure 44D), AD-617747 (Figure 44E), AD-617748 (Figure 44F), and AD-617749 (Figure 44G). [Figure 44B] See legend to Figure 44A. [Figure 44C] See legend to Figure 44A. [Figure 44D] See legend to Figure 44A. [Figure 44E] See legend to Figure 44A. [Figure 44F] See legend to Figure 44A. [Figure 44G] See legend to Figure 44A. [Figure 45] 1 is a line graph showing the time course of serum mTTR levels after administration of a 1 mg / kg dose of exemplary mTTR-targeting duplexes compared to pre-dose. The sense strand of the duplexes contains an LNA-morpholino at position 1 (S1), differing in the number and location of mutations on the PS, and has 5' vinyl phosphate (5'-VP) modification of the antisense strand. [Figure 46] 1 is a bar graph showing the fold change in mTTR gene expression in mouse liver 28 days after administration of exemplary mTTR-targeting duplexes at a dose of 1 mg / kg, where the sense strand of the duplexes contains an LNA-morpholino at position 1 (S1), differing in the number and location of mutations on the PS, and 5'-VP modification of the antisense strand. [Figure 47]

[0023] Figure 1 is a line graph showing the time course of serum mTTR levels after administration of a 1 mg / kg dose of a control mTTR-targeting duplex compared to pre-dose. The sense strand of the duplex contains an LNA at position 1 (S1), differing in the number and location of mutations on the PS, and 5'-VP modification of the antisense strand. DETAILED DESCRIPTION OF THE INVENTION

[0161] Detailed Description It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. As used herein, the use of the singular includes the plural unless specifically stated otherwise. The use of "or" herein means "and / or" unless specifically stated otherwise. Furthermore, the use of the term "including" and other forms, such as "includes" and "included," is not limiting. Furthermore, terms such as "element" or "component" include both elements and components that include one unit and elements and components that include two or more subunits, unless specifically stated otherwise.

[0162] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. Any documents or portions of documents referred to in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for all purposes.

[0163] X S In some embodiments of the various aspects described herein, X S can be O, CH, S, or NH. For example, X S can be O or CH. In some preferred embodiments of any one of the aspects described herein, X S is O.

[0164] R 5 In some embodiments of the various aspects described herein, R 5 Ha-L 1 -R H or -ON(R 13 )R 14 where L 1 is a bond, -L 3 -, C 1~30 Alkylene, C 2~30 Alkenylene, C 2~30 Alkynylene, *-L 3 -C 1~30 Alkylene *-L 3 -C 2~30 Alkenylene, or *-L 3 -C 2~30 alkynylene, and L 3 -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(X L3 )(Y L3 R L3B )- and R L3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or nitrogen protecting group, X L2 is O or S, and Y L3 is O, S, NH, or a bond, and R L3B is H or optionally substituted alkyl, and * is R H is a bond to R H is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally the heterocyclyl contains at least one nitrogen atom or R H teeth TIFF2025534336000046.tif16128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylamino alkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars, and R 13 and R 14 is independently -L 2 -R H2 where L 2 is the linker and R H2 is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, wherein the heterocyclyl is optionally substituted with 1, 2, 3 or 4 independently selected substituents, and optionally R 13 and R 14 At least one of them is -L 2 -R H2 is.

[0165] In some embodiments of any one of the aspects described herein, R 5 Ha-L 1 -R H is.

[0166] In some embodiments of any one of the above aspects, R 5 HA-ON(R 13 )R 14 R 5 -ON(R 13 )R 14 If R 13 and R 14 Note that R can be the same or different. Thus, in some embodiments of any one of the aspects described herein, R 13 and R 14In some embodiments of any one of the aspects described herein, R 13 and R 14 is different.

[0167] In various embodiments described herein, R 13 and R 14 One or both of the -L 2 -R H2 It can be.

[0168] In some embodiments of any one of the aspects described herein, R 13 and R 14 At least one (e.g., one or both) of the groups is -(CH2) m -R H2 or The file is TIFF2025534336000047.tif27128.

[0169] In some embodiments of any one of the aspects described herein, R 5 is N3.

[0170] In some embodiments of any one of the aspects described herein, R 5 teeth TIFF2025534336000048.tif80159TIFF2025534336000049.tif185159, where n is 0 or an integer selected from 1-30 (e.g., 1-20, e.g., 1, 2, 3, 4, 5, or 6), X is ONH, S, or CH2, and L is a ligand or a linker covalently linked to one or more ligands (e.g., L is an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar).

[0171] In some embodiments of any one of the aspects described herein, R 5 teeth TIFF2025534336000050.tif21136.

[0172] R 3 In some embodiments of any one of the aspects described herein, R 3 is a reactive phosphorus group, hydrogen, halogen, -OR 232 , -SR 233 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 234 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 235 , NHC(O)R 236 , a lipid, a linker covalently bonded to a lipid, a ligand, or a linker covalently bonded to a ligand.

[0173] R 232 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 233 is H, a sulfur protecting group, an optionally substituted C 1~30 Alkyl, C 1~30Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 234 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 235 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, amino (NH), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl. 236 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 It can be alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.

[0174] In some embodiments of any one of the aspects described herein, R 3 is a reactive phosphorus group.

[0175] Without wishing to be bound by theory, reactive phosphorus groups are useful for forming internucleoside linkages, including, for example, phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and include, but are not limited to, phosphoramidites, H-phosphonates, phosphate triesters, and phosphorus-containing chiral auxiliary groups. III or P V Contains phosphorus atoms in the valence state. Phosphoramidites (P III Reactive phosphorus(III) groups in the form of (III)-( ...

[0176] In some embodiments of any one of the aspects described herein, the reactive phosphorus(III) group is —OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )RP3 For example, a reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2).

[0177] In some embodiments of any one of the above aspects, R P is optionally substituted C 1~6 alkyl. For example, R P are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 1~6 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. In some embodiments, R p may be substituted by CN or -SC(O)Ph, C 1~6 alkyl. For example, R p is cyanoethyl (-CH2CH2CN).

[0178] In the reactive phosphorus(III) group, each R P2 are independently optionally substituted C 1~6 For example, each RP2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl. P2 It should be noted that when groups are present, they can be the same or different. Thus, in some non-limiting examples, two or more R P2 If groups are present, then those R P2 In some other non-limiting examples, two or more R P2 If groups are present, then those R P2 In some embodiments of any one of the above aspects, each R P2 is isopropyl.

[0179] In some embodiments of any one of the above aspects, both R P2taken together with the nitrogen atom to which they are attached form an optionally substituted 3- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, and the like, each of which is optionally substituted with OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C (1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2 or 3 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0180] In some embodiments of any one of the above aspects, R P and R P2and one of the following, taken together with the atom to which they are attached, forms an optionally substituted 4- to 8-membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolidinyl, 1,4-diazaperhydroepynyl, 1,3-dioxanyl, 1,4-dioxanyl, and the like, each of which is optionally substituted with OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C (1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p It may be substituted with 1, 2 or 3 substituents independently selected from -NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5 or 6.

[0181] In the reactive phosphorus(III) group, each R P3 are independently optionally substituted C 1~6 alkyl. For example, R P3are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 1~6 alkyl, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R P3 is methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, pentyl, or hexyl, each of which is optionally substituted with NH, OH, C(O)NH, COOH, halo, SH, or C-C alkoxy.

[0182] In some embodiments of any one of the above aspects, the reactive phosphorus(III) group is —OP(OR P )(N(R P2 )2). For example, the reactive phosphorus(III) group is -OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0183] In some embodiments of any one of the aspects described herein, R 3 -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 where each R P is cyanoethyl (-CH2CH2CN), and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0184] In some embodiments of any one of the above aspects, R 3 -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P ) optionally substituted C 1~6 alkyl, where each R Pis cyanoethyl (-CH2CH2CN), and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0185] In some embodiments of any one of the above aspects, R 3 -OP(OR P )(N(R P2 )2). For example, R 3 -OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0186] In some embodiments of any one of the above aspects, R 3 -OR 232 If R 232 can be hydrogen or a hydroxyl protecting group. For example, R 232 In some embodiments of any one of the aspects described herein, R can be hydrogen. 23 is -OC(O)CH2CH2CO2H.

[0187] R 3 Ga-SR 233 If R 233 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R 233 is hydrogen.

[0188] R 3 -O(CH2CH2O) r CH2CH2OR 234 where r can be 1 to 50, and R 234 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R235 and R 235 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0189] R 3 But -NH(CH2CH2NH) s CH2CH2-R 235 where s can be 1 to 50, and R 235 can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0190] In some embodiments of any one of the aspects described herein, R 3 is hydrogen, halogen, -OR 232 or optionally substituted C1 to C 30 Alkoxy. For example, R 3 is halogen, -OR 232 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R 3 is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0191] In some embodiments of any one of the aspects described herein, R 3are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 23 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R 23 is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0192] In some embodiments of any one of the above aspects, R 3 is -O(CH2) u R 237 where u is 2 to 10, and R 237 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R 237is —CH or —NH. Thus, in some embodiments of any one of the aspects described herein, R 3 is -O(CH2) u -OMe or R 23 is -O(CH2) u It is NH2.

[0193] In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0194] In some embodiments of any one of the aspects described herein, R 3 is C1-C6 haloalkyl. For example, R 3 is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R 23 is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0195] In some embodiments of any one of the aspects described herein, R 3 -OCH(CH2OR 238 )CH2OR 239 where R 238 and R 239 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 238 and R 239 are independently optionally substituted C1 to C 30 It is alkyl. In some embodiments of any one of the aspects described herein, R 23 -CH2C(O)NHR 2310 where R 2310 is H, optionally substituted C1 to C 30Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 2310 is H or optionally substituted C1 to C 30 In some embodiments, R 2310 is an optionally substituted C1-C6 alkyl.

[0196] R 2 In some embodiments of any one of the aspects described herein, R 2 is hydrogen, halogen, -OR 222 , -SR 223 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 224 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 225 , NHC(O)R 226 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, or a reactive phosphorus group.

[0197] R 222 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 223 is H, a sulfur protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 224 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 225 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, amino (NH), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl. 226 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30It can be alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.

[0198] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, halogen, -OR 222 , -SR 223 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 224 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 225 , NHC(O)R 224 is.

[0199] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -OC 4~30 Alkyl-ON(CH2R 8)(CH2R 9 ), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9 ) For example, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 It is alkoxy, alkoxyalkyl (eg, methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.

[0200] In some embodiments of any one of the above aspects, R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 Alkoxy, or alkoxyalkyl (e.g., methoxyethyl. In some embodiments of any one of the above aspects, R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro or methoxy.

[0201] In some embodiments of any one of the above aspects, R 2 is a halogen. For example, R 2 can be fluoro, chloro, bromo, or iodo. In some embodiments of any one of the aspects described herein, R 2 is fluoro.

[0202] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, fluoro or methoxy.

[0203] In some embodiments of any one of the above aspects, R 2 -OR 222 If R 222 can be hydrogen or a hydroxyl protecting group.

[0204] R 2 Ga-SR 223 If R 223can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R 223 is hydrogen.

[0205] R 2 -O(CH2CH2O) r CH2CH2OR 224 where r can be 1 to 50, and R 224 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 225 and R 225 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0206] R 2 But -NH(CH2CH2NH) s CH2CH2-R 225 where s can be 1 to 50, and R 225 can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0207] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, halogen, -OR 222 or optionally substituted C1 to C 30 Alkoxy. For example, R 2 is halogen, -OR 222 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R 2 is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0208] In some embodiments of any one of the aspects described herein, R 2 are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 22 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0209] In some embodiments of any one of the above aspects, R 2 is -O(CH2) u R 227 where u is 2 to 10, and R 227is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R 227 is —CH or NH. Thus, in some embodiments of any one of the aspects described herein, R 2 is -O(CH2) u -OMe or R 2 is -O(CH2) u In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0210] In some embodiments of any one of the aspects described herein, R 2 is C1-C6 haloalkyl. For example, R 2 is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R 2 is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0211] In some embodiments of any one of the aspects described herein, R 2 -OCH(CH2OR 228 )CH2OR 229 where R 228 and R 229 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 228 and R 229 are independently optionally substituted C1 to C 30 It is alkyl.

[0212] In some embodiments of any one of the aspects described herein, R 2 -CH2C(O)NHR 2210 where R 2210is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 2210 is H or optionally substituted C1-C 30 In some embodiments, R 2210 is an optionally substituted C1-C6 alkyl.

[0213] In some embodiments of any one of the above aspects, R 2 -OR 222 If R 222 can be hydrogen or a hydroxyl protecting group.

[0214] R 2 Ga-SR 223 If R 223 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R 223 is hydrogen.

[0215] R 2 -O(CH2CH2O) r CH2CH2OR 224 where r can be 1 to 50, and R 224 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 225 and R 225 is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0216] R 2 But -NH(CH2CH2NH) s CH2CH2-R 225 where s can be 1 to 50, and R 225can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0217] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, halogen, -OR 222 or optionally substituted C1 to C 30 Alkoxy. For example, R 2 is halogen, -OR 222 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R 2 is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0218] In some embodiments of any one of the aspects described herein, R 2 are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 22 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R 2 is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0219] In some embodiments of any one of the above aspects, R 2 is -O(CH2) u R 227 where u is 2 to 10, and R 227 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R 227 is —CH or NH. Thus, in some embodiments of any one of the aspects described herein, R 2 is -O(CH2) u -OMe or R 2 is -O(CH2) u In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0220] In some embodiments of any one of the aspects described herein, R 2 is C1-C6 haloalkyl. For example, R 2 is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R 2 is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0221] In some embodiments of any one of the aspects described herein, R 2 -OCH(CH2OR 228 )CH2OR 229 where R 228 and R 229 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 228 and R 229 are independently optionally substituted C1 to C 30 It is alkyl.

[0222] In some embodiments of any one of the aspects described herein, R 2 -CH2C(O)NHR 2210 where R 2210 is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 2210 is H or optionally substituted C1-C 30 In some embodiments, R 2210 is an optionally substituted C1-C6 alkyl.

[0223] In some embodiments of any one of the aspects described herein, R 2 is a reactive phosphorus group. For example, R 2 -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(NR P2 )2, -OP(O)(ORP )H, -OP(S)(OR P )H, -OP(O)(SR P )H, -OP(O)(OR P )R P3 , -OP(S)(OR P )R P3 , or -OP(O)(SR P )R P3 where each R P is cyanoethyl (-CH2CH2CN), and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0224] In some embodiments of any one of the above aspects, R 2 -OP(OR P )(N(R P2 )2), -OP(SR P )(N(R P2 )2), -OP(O)(OR P )(N(R P2 )2), -OP(S)(OR P )(N(R P2 )2), -OP(O)(SR P )(N(R P2 )2), -OP(O)(OR P )H, -OP(S)(OR P ) optionally substituted C 1~6 alkyl, where each R P is cyanoethyl (-CH2CH2CN), and each R P2 are independently optionally substituted C 1~6 alkyl, and each R P3 are independently optionally substituted C 1~6 It is alkyl.

[0225] In some embodiments of any one of the above aspects, R 2 -OP(OR P )(N(R P2 )2). For example, R 2-OP(OR P )(N(R P2 )2), where R P is cyanoethyl (-CH2CH2CN), and each R P2 is isopropyl.

[0226] R 2 and R 3 Note that only one of R can be a reactive phosphorus group. 3 is a phosphorus(III) group.

[0227] In some embodiments of any one of the aspects described herein, R 2 and R 4 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v -2', v is 1, 2 or 3, and Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )- and R 10 and R 11 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, and R 12 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alky-CO2H, or nitrogen protecting group.

[0228] In some embodiments of any one of the above aspects, v is 1. In some other embodiments of any one of the above aspects, v is 2.

[0229] In some embodiments, Y is O. For example, R 2 and R 4 together form 4'-C(R 10 R 11 ) v -O-2'.

[0230] R attached to the same carbon 10 and R 11 Note that R can be the same or different. For example, R 10 and R 11 can be H, and one of R 10 and R 11 The other of R can be an optionally substituted C1-C6 alkyl. 10 and R 11 One of the groups can be H and the other can be OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl(s) i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) pand C1-C6 alkyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 10 and R 11 are independently H or C1-C optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy. 30 In some embodiments of any one of the above aspects, R 10 and R 11 One of them is H, and the other is C1-C6 alkyl optionally substituted with C1-C6 alkoxy. For example, R 10 and R 11 One of them is H and the other is —CH3 or CH2OCH3.

[0231] In some embodiments of any one of the above aspects, R 10 and R 11 For example, R attached to the same C 10 and R 11 is H.

[0232] In some embodiments of any one of the above aspects, R 2 and R 4 taken together are 4'-CH2-O-2', 4'-CH(CH3)-O-2', 4'-CH(CH2OCH3)-O-2', or 4'-CH2CH2-O-2'. For example, R 2 and R 4 together form 4'-CH2CH2-O-2'.

[0233] In some embodiments of any one of the aspects described herein, R 2 and R 4 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 )v -2' and R 3 is a reactive phosphorus(III) group, a hydroxyl or a protected hydroxyl.

[0234] In some embodiments of any one of the aspects described herein, R 2 is hydrogen, fluoro or methoxy, and R 3 is a reactive phosphorus(III) group, hydroxyl or protected hydroxyl, and R 4 is H.

[0235] R 4 In some embodiments of any one of the aspects described herein, R 4 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 For example, R 4 is hydrogen, optionally substituted C 1~6 Alkyl or optionally substituted C 1~6 It can be an alkoxy.

[0236] In some embodiments of any one of the aspects described herein, R 4 is H.

[0237] R 23 In some embodiments of any one of the aspects described herein, R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, a hydroxyl, a protected hydroxyl, an optionally substituted C 1~30Alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), amino, alkylamino, dialkylamino, a 3'-oligonucleotide capping group (e.g., an inverted nucleotide or an inverted abasic nucleotide), a ligand, or a linker covalently attached to one or more ligands (e.g., N-acetylgalactosamine (GalNac)).

[0238] In some embodiments of any one of the aspects described herein, R 23 is the bond to the internucleotide linkage to the subsequent nucleotide. 23 and R 22 Note that only one of R can be the bond to the internucleotide linkage to the subsequent nucleotide. 23 is the bond to the internucleotide linkage to the subsequent nucleotide.

[0239] In some embodiments of any one of the aspects described herein, R 23 is hydrogen, halogen, -OR 232 , -SR 233 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 234 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 235 , NHC(O)R 236 , a lipid, a linker covalently bonded to a lipid, a ligand, or a linker covalently bonded to a ligand.

[0240] R 232 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 233 is H, a sulfur protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 234 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 235 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, amino (NH), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl. 236is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 It can be alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.

[0241] In some embodiments of any one of the above aspects, R 23 -OR 232 If R 232 can be hydrogen or a hydroxyl protecting group. For example, R 232 can be hydrogen, a hydroxyl protecting group, or an alkyl group (e.g., methoxy) in some embodiments of any one of the aspects described herein.

[0242] R 23 Ga-SR 233 If R 233 can be hydrogen or a sulfur protecting group. Thus, in some embodiments of any one of the above aspects, R 233 is hydrogen.

[0243] R 23 -O(CH2CH2O) r CH2CH2OR 234 where r can be 1 to 50, and R 234 are independently generated for each occurrence, H, C1 to C 30 alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R 235 and R 235is independently at each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0244] R 23 But -NH(CH2CH2NH) s CH2CH2-R 235 where s can be 1 to 50, and R 235 can be, independently at each occurrence, amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroarylamino.

[0245] In some embodiments of any one of the aspects described herein, R 23 is hydrogen, halogen, -OR 232 or optionally substituted C1 to C 30 Alkoxy. For example, R 23 is halogen, -OR 232 or optionally substituted C1 to C 30 In some embodiments of any one of the aspects described herein, R 23 is F, OH or optionally substituted C1-C 30 It is an alkoxy.

[0246] In some embodiments of any one of the aspects described herein, R 23are OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl (i.e., C1-C8 alkyl). koxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p C1-C optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy 30 alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 23 is optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy, 30 In some embodiments of any one of the aspects described herein, R 23 is -O(CH2) t CH3, where t is 1 to 21. For example, t is 14, 15, 16, 17, or 18. In one non-limiting example, t is 16.

[0247] In some embodiments of any one of the above aspects, R 23 is -O(CH2) u R 237 where u is 2 to 10, and R 237 is C1-C6 alkoxy, amino (NH2), CO2H, OH, or halo. For example, R 237is —CH or —NH. Thus, in some embodiments of any one of the aspects described herein, R 23 is -O(CH2) u -OMe or R 23 is -O(CH2) u In some embodiments of any one of the aspects described herein, u is 2, 3, 4, 5, or 6. For example, u is 2, 3, or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0248] In some embodiments of any one of the aspects described herein, R 23 is C1-C6 haloalkyl. For example, R 23 is C1-C4 haloalkyl. In some embodiments of any one of the aspects described herein, R 23 is -CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0249] In some embodiments of any one of the aspects described herein, R 23 -OCH(CH2OR 238 )CH2OR 239 where R 238 and R 239 are independently H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C 30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 238 and R 239 are independently optionally substituted C1 to C 30 It is alkyl.

[0250] In some embodiments of any one of the aspects described herein, R 23 -CH2C(O)NHR 2310 where R 2310 is H, optionally substituted C1 to C 30 Alkyl, optionally substituted C2-C30 Alkenyl or optionally substituted C2-C 30 Alkynyl. For example, R 2310 is H or optionally substituted C1 to C 30 In some embodiments, R 2310 is an optionally substituted C1-C6 alkyl.

[0251] R 22 In some embodiments of any one of the aspects described herein, R 22 is hydrogen, halogen, -OR 222 , -SR 223 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 224 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 225 , NHC(O)R 226 , a lipid, a linker covalently attached to a lipid, a ligand, a linker covalently attached to a ligand, or a reactive phosphorus group.

[0252] R 222 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 223 is H, a sulfur protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 224 is H, a hydroxyl protecting group, an optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 225 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 R can be alkoxy, amino (NH), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl. 226 is hydrogen, halogen, hydroxyl, protected hydroxyl, optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30It can be alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, or heteroaryl.

[0253] In some embodiments of any one of the aspects described herein, R 22 is hydrogen, halogen, -OR 222 , -SR 223 , optionally substituted C 1~30 Alkyl, C 1~30 Haloalkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, or optionally substituted C 1~30 Alkoxy, amino (NH2), alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, amino acid, -O(CH2CH2O) r CH2CH2OR 224 , cyano, alkyl-thio-alkyl, thioalkoxy, cycloalkyl, aryl, heteroaryl, -NH(CH2CH2NH) s CH2CH2-R 225x , NHC(O)R 224 is.

[0254] In some embodiments of any one of the aspects described herein, R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 Alkyl, optionally substituted C 2~30 Alkenyl, optionally substituted C 2~30 Alkynyl, optionally substituted C 1~30 Alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -OC 4~30 Alkyl-ON(CH2R 8)(CH2R 9 ), or -OC 4~30 Alkyl-ON(CH2R 8 )(CH2R 9x For example, R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 It is alkoxy, alkoxyalkyl (eg, methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, or dialkylamino.

[0255] In some embodiments of any one of the above aspects, R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1~30 Alkoxy, or alkoxyalkyl (e.g., methoxyethyl. In some embodiments of any one of the above aspects, R 22 is hydrogen, hydroxyl, protected hydroxyl, fluoro or methoxy.

[0256] In some embodiments of any one of the above aspects, R 22 is a halogen. For example, R 22 can be fluoro, chloro, bromo, or iodo. In some embodiments of any one of the aspects described herein, R 22 is fluoro.

[0257] In some embodiments of any one of the aspects described herein, R 22 is hydrogen, fluoro or methoxy.

[0258] In some embodiments of any one of the aspects described herein, R 22 and R 24 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC(R 10 R 11 ) v-2', v is 1, 2 or 3, and Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O)2-, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 )- and R 10 and R 11 are independently H, optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl, and R 12 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alky-CO2H, or a nitrogen protecting group. In some embodiments of any one of the above aspects, v is 1. In other embodiments of any one of the above aspects, v is 2. In some embodiments, Y is O. For example, R 2 and R 4 together form 4'-C(R 10 R 11 ) v -O-2'.

[0259] R attached to the same carbon 10 and R 11 Note that R can be the same or different. For example, R 10 and R 11 can be H, and one of R 10 and R 11 The other of R can be an optionally substituted C1-C6 alkyl. 10 and R 11One of the groups can be H and the other can be OH, CN, SC(O)Ph, oxo(=O), SH, SO2NH2, SO2(C1-C4)alkyl, SO2NH(C1-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (C1-C8)alkyl, O(C1-C8)alkyl(s) i.e., C1-C8 alkoxy), O(C1-C8)haloalkyl, (C2-C8)alkenyl, (C2-C8)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2-C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2-C(O)-alkyl, C(O)-alkyl, alkylcarbonylaminyl, CH2-[CH(OH)] m -(CH2) p -OH, CH2-[CH(OH)] m -(CH2) p and C1-C6 alkyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from —NH2 or CH2-aryl-alkoxy, where "m" and "p" are independently 1, 2, 3, 4, 5, or 6. For example, R 10 and R 11 are independently H or C1-C optionally substituted with NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6 alkoxy. 30 In some embodiments of any one of the above aspects, R 10 and R 11 One of them is H, and the other is C1-C6 alkyl optionally substituted with C1-C6 alkoxy. For example, R 10 and R 11 One of them is H and the other is —CH3 or CH2OCH3.

[0260] In some embodiments of any one of the above aspects, R 10 and R 11 For example, R attached to the same C10 and R 11 is H.

[0261] In some embodiments of any one of the above aspects, R 22 and R 24 taken together are 4'-CH2-O-2', 4'-CH(CH3)-O-2', 4'-CH(CH2OCH3)-O-2', or 4'-CH2CH2-O-2'. For example, R 22 and R 24 together form 4'-CH2CH2-O-2'.

[0262] In some embodiments of any one of the aspects described herein, R 22 is the bond to the internucleotide linkage to the subsequent nucleoside.

[0263] R 24 In some embodiments of any one of the aspects described herein, R 24 is hydrogen, optionally substituted C 1~6 Alkyl, optionally substituted C 2~6 Alkenyl, optionally substituted C 2~6 Alkynyl, or optionally substituted C 1~6 For example, R 24 is hydrogen, optionally substituted C 1~6 Alkyl or optionally substituted C 1~6 It can be an alkoxy.

[0264] In some embodiments of any one of the aspects described herein, R 24 is H.

[0265] L 1 In some embodiments of any one of the aspects described herein, L 1 can be a linker.

[0266] For example, L 1is a direct bond or an atom such as oxygen or sulfur, NR LL , C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH, or a chain of atoms, such as a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, where one or more methylenes are O, S, S(O), SO2, N(R LL )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group, wherein R LL is hydrogen, acyl, aliphatic or substituted aliphatic.

[0267] In some embodiments of any one of the aspects described herein, L 1 is a bond or an optionally substituted alkylene. For example, L 1 is a bond. In some other non-limiting examples, L 1 is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R LL )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group.

[0268] In some embodiments of any one of the aspects described herein, L 1 is a bond, -L 3 -, C 1~30 Alkylene, C 2~30 Alkenylene, C 2~30 Alkynylene, *-L 3 -C 1~30 Alkylene *-L 3 -C 2~30 Alkenylene, or *-L 3 -C 2~30 It is alkynylene.

[0269] In some embodiments of any one of the aspects described herein, L 1 is L 3 where L 3 -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(X L3 )(Y L3 R L3B )-, where R L3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4 Alkynyl, optionally substituted C 1~30 alkyl-COH, or nitrogen protecting group, X L2 is O or S, and Y L3 is O, S, NH, or a bond, and R L3B is H or optionally substituted alkyl. In some embodiments, L 3 is -O-.

[0270] In some embodiments of any one of the aspects described herein, L 1 is C1 ~30 Alkylene, C 2~30 Alkenylene, C 2~30 Alkynylene, *-L 3 -C 1~30 Alkylene *-L 3 -C 2~30 Alkenylene, or *-L 3 -C 2~30 alkynylene, where * is R H is a bond to L 3 is R L3 is hydrogen, optionally substituted C 1~30 Alkyl, optionally substituted C1-C 30 Alkoxy, C 1~4 Haloalkyl, optionally substituted C 2~4 Alkenyl, optionally substituted C 2~4Alkynyl, optionally substituted C 1~30 alkyl-COH, or nitrogen protecting group, X L2 is O or S, and Y L3 is O, S, NH, or a bond, and R L3B is H or optionally substituted alkyl.

[0271] In some embodiments of the various aspects described herein, L 1 is a bond, —O—, or an optionally substituted alkylene. For example, L 1 is -O- or -(CH2) n -wherein n is 0 or an integer selected from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., n is 1, 2, 3, 4, 5, or 6).

[0272] In some embodiments of any one of the aspects described herein, L 1 In some other embodiments of any one of the aspects described herein, L 1 is methylene, i.e., —CH—. In still some other embodiments of any one of the aspects described herein, L 1 is a bond.

[0273] In some embodiments of any one of the aspects described herein, L 1 teeth TIFF2025534336000051.tif17128, where b' is 0 or an integer from 1 to 20 (e.g., b' is 0, 1, 2, 3, 4, 5, or 6), and # is R H For example, L 1 teeth TIFF2025534336000052.tif24128.

[0274] L 2 In some embodiments of any one of the aspects described herein, L 2is a linker. For example, L 2 is a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH, or a chain of atoms, such as a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, where one or more methylenes are O, S, S(O), SO2, N(R LL )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group, wherein R LL is hydrogen, acyl, aliphatic or substituted aliphatic.

[0275] In some embodiments of any one of the aspects described herein, L 2 is a bond or an optionally substituted alkylene. For example, L 2 is a bond. In some other non-limiting examples, L 2 is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R LL )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group. 2 -Z-(CH2) m -, where Z is absent or is aryl, heteroaryl, cycloalkyl, or heterocyclyl, and m is 0 or an integer selected from 1 to 20 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., m is 1, 2, 3, 4, 5, or 6). For example, L 2 Ha-(CH2) m -or-(CH2) m -phenyl-.

[0276] L 3 In various embodiments described herein, L 3 -O-, -N(R L3 )-, -S-, -C(O)-, -S(O)-, -S(O)2-, -P(X L3 )(Y L3 R L3B )-. For example, L 3 In some embodiments of any one of the aspects described herein, L can be -O-. 3 is -N(R L3 In some further embodiments of any one of the aspects described herein, L 3 is -N(R L3 In still some other embodiments of any one of the aspects described herein, L 3 -P(X L3 )(Y L3 R L3B )- can be.

[0277] R H In some embodiments of any one of the aspects described herein, R H is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S. For example, R H teeth TIFF2025534336000053.tif13128, where X is O, NR L , S, or CH2, and R Lis hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0278] In some embodiments of any one of the aspects described herein, R H teeth TIFF2025534336000054.tif13128, where X is O.

[0279] In some other embodiments of any one of the aspects described herein, R H teeth TIFF2025534336000055.tif13128, where X is NR L In some further embodiments, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. H is a ligand or a linker covalently attached to one or more independently selected ligands.

[0280] In some embodiments of any one of the aspects described herein, R H teeth TIFF2025534336000056.tif13128, where X is O.

[0281] In some other embodiments of any one of the aspects described herein, R H teeth TIFF2025534336000057.tif13128, where X is NR L In some further embodiments, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0282] R H2 In some embodiments of any one of the aspects described herein, R H2 is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1, or 2 additional heteroatoms independently selected from N, O, and S. For example, R H2 teeth TIFF2025534336000058.tif13128, where X is O, NR L , S, or CH2, and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar.

[0283] In some embodiments of any one of the aspects described herein, R H2 teeth TIFF2025534336000059.tif13128, where X is O.

[0284] In some other embodiments of any one of the aspects described herein, R H2 teeth TIFF2025534336000060.tif13128, where X is NR L In some further embodiments, R L is H or an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. L is a ligand or a linker covalently attached to one or more independently selected ligands.

[0285] In some embodiments of any one of the aspects described herein, R 13 and R 14 One of the groups is an optionally substituted C1-C6 alkyl. For example, R 13 and R 14 One of the groups is methyl.

[0286] In some embodiments of any one of the aspects described herein, R 13 and R 14 One of them is -L 2 -R H2 and the other is an optionally substituted C1-C6 alkyl (for example, methyl).

[0287] In some embodiments of any one of the aspects described herein, R 13 and R 14 One of the TIFF2025534336000061.tif22128, R 13 and R 14 the other is C1-C6 alkyl, TIFF2025534336000062.tif22128.

[0288] R L In various embodiments described herein, R L can be hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, an alkyl ester, an alkylamine, a dimethylamino alkyl, an alkyl ether, an alkyl thioether, a heteroaromatic alkyl, an allyl, a vinyl, a disulfide, an oxime, a ketone, an acetal, a hemiacetal, a cleavable peptide, or a cleavable sugar.

[0289] In some embodiments of any one of the aspects described herein, R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, or an optionally substituted aliphatic. For example, R L is a linker covalently attached to the ligand, one or more ligands.

[0290] In some embodiments of any one of the aspects described herein, R L Ha-L 4 -L R where L 4 is a linker and L R is a ligand, a linker covalently attached to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar. For example, L R is the ligand.

[0291] In some embodiments of any one of the aspects described herein, L Ris an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylaminoalkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars. For example, L R is C 1~30 Alkyl, C 2~30 Alkenyl, C 2~30 The ligand may be an alkynyl, lipid, carbohydrate, folate, DUPA, RGD peptide, antibody, antibody fragment, peptide or other ligand.

[0292] In some embodiments of any one of the aspects described herein, L 4 is a direct bond or an atom such as oxygen or sulfur, NR LL , C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH, or a chain of atoms, such as a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, where one or more methylenes are O, S, S(O), SO2, N(R LL )2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group, wherein R LL is hydrogen, acyl, aliphatic or substituted aliphatic.

[0293] In some embodiments of any one of the aspects described herein, L 4 is a bond or an optionally substituted alkylene. For example, L 4 is a bond. In some other non-limiting examples, L 4 is a substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, or substituted or unsubstituted alkynyl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R LL)2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group. 4 -Z-(CH2) m -, where Z is absent or is aryl, heteroaryl, cycloalkyl, or heterocyclyl, and m is 0 or an integer selected from 1 to 20 (e.g., m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, e.g., m is 1, 2, 3, 4, 5, or 6). For example, L 4 Ha-(CH2) m -or-(CH2) m -phenyl-.

[0294] In some embodiments of any one of the aspects described herein, L 4 teeth TIFF2025534336000063.tif16128, where c' is 0 or an integer from 1 to 20 (e.g., d' is 0, 1, 2, 3, 4, 5, or 6). For example, L 4 teeth In some embodiments, c' is 1.

[0295] In some embodiments of any one of the aspects described herein, R L teeth TIFF2025534336000065.tif17128, where d' is 0 or an integer between 1 and 20 (e.g., d' is 0, 1, 2, 3, 4, 5, or 6). In some embodiments, d' is 1.

[0296] In some embodiments of any one of the aspects described herein, R L teeth TIFF2025534336000066.tif16128, where d' is 0 or an integer between 1 and 20 (e.g., d' is 0, 1, 2, 3, 4, 5, or 6). For example, R L teeth TIFF2025534336000067.tif24128. In some embodiments, d' is 1.

[0297] In some embodiments of any one of the aspects described herein, R L Ha-C(O)-L R is.

[0298] In some embodiments of any one of the aspects described herein, R L is a nitrogen protecting group.

[0299] B (nucleobase) In various aspects described herein, B is an optionally modified nucleobase. Note that the nucleobase can be a natural nucleobase or an unnatural nucleobase. "Unnatural nucleobase" refers to a nucleobase other than adenine, guanine, cytosine, uracil, or thymine. Exemplary unnatural nucleobases include inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, and substituted or modified analogs of adenine, guanine, cytosine, and uracil, such as 2-aminoadenine and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil, and and 5-halocytosine, 5-propynyluracil and 5-propynylcytosine, 6-azouracil, 6-azocytosine and 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-aminoallyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and 5- Substituted cytosine, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, such as 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkylcytosine, 7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine, 5-amino- aryl-uracil, N3-methyluracil, substituted 1,2,4-triazole, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N 4

[0023] Additional purines and pyrimidines include, but are not limited to, 2-acetylcytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanine, or O-alkylated bases. Additional purines and pyrimidines include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Concise Encyclopedia of Polymer Science and Engineering, edited by Kroschwitz, JI, John Wiley & Sons, 1990, pages 858-859, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613, the contents of all of which are incorporated herein by reference.

[0300] In some embodiments, the unnatural nucleobase is inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidin, 2-(halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyl)adenine, 2-(aminopropyl)adenine, 2-(methylthio)-N 6 -(Isopentenyl)adenine, 7-(Deaza)adenine, 8-(Alkenyl)adenine, 8-(Alkyl)adenine, 8-(Alkynyl)adenine, 8-(Amino)adenine, 8-(Halo)adenine, 8-(Hydroxyl)adenine, 8-(Thioalkyl)adenine, 8-(Thiol)adenine, N 6 -(Isopentyl)adenine, N 6 -(methyl)adenine, N 6 ,N 6-(Dimethyl)adenine, 2-(alkyl)guanine, 2-(propyl)guanine, 6-(alkyl)guanine, 6-(methyl)guanine, 7-(alkyl)guanine, 7-(methyl)guanine, 7-(deaza)guanine, 8-(alkyl)guanine, 8-(alkenyl)guanine, 8-(alkynyl)guanine, 8-(amino)guanine, 8-(halo)guanine, 8-(hydroxyl)guanine, 8-(thioalkyl)guanine cytosine, 8-(thiol)guanine, N-(methyl)guanine, 2-(thio)cytosine, 3-(deaza)-5-(aza)cytosine, 3-(alkyl)cytosine, 3-(methyl)cytosine, 5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5-(halo)cytosine, 5-(methyl)cytosine, 5-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N 4 -(Acetyl)cytosine, 3-(3-amino-3-carboxypropyl)uracil, 2-(thio)uracil, 5-(methyl)-2-(thio)uracil, 5-(methylaminomethyl)-2-(thio)uracil, 4-(thio)uracil, 5-(methyl)-4-(thio)uracil, 5-(methylaminomethyl)-4-(thio)uracil, 5-(methyl)-2,4-(dithio)uracil, 5-(methylaminomethyl)-2,4-(dithio)uracil, 5-(2-aminopropyl)uracil, 5-(alkyl)uracil, 5-(alkynyl)uracil, 5-(allylamino)uracil, 5-(aminoallyl)uracil , 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(methoxycarbonylmethyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N 3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil, 4-(thio)pseudouracil, 2,4-(dithio)pseudouracil, 5-(alkyl)pseudouracil, 5-(methyl)pseudouracil, 5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil, 5-(alkyl)-4-(thio)pseudouracil, 5-(methyl)-4-(thio)pseudouracil, 5-(alkyl)-2,4-(dithio)pseudouracil Uracil, 5-(methyl)-2,4-(dithio)pseudouracil, 1-substituted pseudouracil, 1-substituted 2(thio)-pseudouracil, 1-substituted 4-(thio)pseudouracil, 1-substituted 2,4-(dithio)pseudouracil, 1-(aminocarbonylethylenyl)-pseudouracil, 1-(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1-(aminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil uracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1, 3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl 7-(guanidinium alkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidinium alkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidinium alkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 1,3,5-(triaza)-2,6-(dioxa-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidin, isoguanisine, inosinyl, 2-aza-inosinyl, 7-deaza -Inosinyl, nitroimidazolyl, nitropyrazolyl, nitrobenzimidazolyl, nitroindazolyl, aminoindolyl, pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl, 5-(methyl)isocarbostyrilyl, 3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl, 6-(methyl)-7-(aza)indolyl, imidizopyridinyl, 9-(methyl)-imidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl Indolyl, 2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, naphthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, difluorotolyl, 4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole, 6-(azo)thymine, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 6-(aza)pyrimidine, 2-(amino)purine, 2,6-(diamino)purine, 5-substituted pyrimidines, N, 2-substituted purines, N 6 -substituted purines, O 6 -substituted purines, substituted 1,2,4-triazoles, and any O- or N-alkylated derivatives thereof.

[0301] In some embodiments, non-natural nucleobase is modified nucleobase.That is, nucleobase comprises nucleobase modification as described herein, for example, nucleobase is any substitution or modified analogue of natural nucleobase.Examples of nucleobase modification include C-5 pyrimidine modification with alkyl or amino alkyl and other cationic groups, for example, guanidinium and amidine functional groups, known in the art; N-substituted purine with alkyl or amino alkyl and other cationic groups, for example, guanidinium and amidine functional groups; 2 - and N 6 -modifications, G-clamp, guanidinium G-clamp, and pseudouridine.

[0302] In some embodiments of any one of the above aspects, the unnatural nucleobase is a universal nucleobase. As used herein, a universal nucleobase refers to any modified or unmodified natural or unnatural nucleobase that can base pair with adenine, cytosine, guanine, and uracil without substantially affecting the melting behavior, intracellular enzyme recognition, or activity of the oligonucleotide containing the universal nucleobase. Exemplary universal nucleobases include 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza-7-deazaadenine, 4-fluoro-6-methylbenzimidazole, 4-methylimidazole, 3-methylisocarbostyrilyl, 5-methylisocarbostyrilyl, 3-methyl-7-propynylisocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, and imidizopyridinyl. , 9-methylimidizopyridinyl, pyrrolopyridinyl, isocarbostyrilyl, 7-propynylisocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof.

[0303] In some embodiments of any one of the aspects described herein, the non-natural nucleobase is a protected nucleobase. As used herein, "protected nucleobase" refers to a nucleobase that includes a nitrogen-protecting group, an oxygen-protecting group, and / or a sulfur-protecting group.

[0304] In some embodiments of any one of the aspects described herein, the non-natural nucleobase is a modified, protected, or substituted analog of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.

[0305] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C4 position.

[0306] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C5 position.

[0307] In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the N2 position. In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the N6 position.

[0308] In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the C6 position.

[0309] In some embodiments of any one of the aspects described herein, the nucleobase is an N-7 deazapurine optionally modified at the N7 position.

[0310] double stranded RNA Those skilled in the art are well aware that double-stranded RNAs containing 20-23 base pairs, especially 21 base pairs, are recognized to be particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, other researchers have found that shorter or longer double-stranded oligonucleotides can also be effective.

[0311] Therefore, in one aspect, the present application provides double-stranded RNA (dsRNA), which comprises first strand (also referred to as antisense strand or guide strand) and second strand (also referred to as sense strand or passenger strand), and at least one of the first strand (i.e., antisense strand) or second strand (i.e., sense strand) is the oligonucleotide described herein.In other words, at least one of the first strand (i.e., antisense strand) or second strand (i.e., sense strand) comprises at least one nucleotide of formula (II).

[0312] In some embodiments of any one of the aspects described herein, the sense strand is an oligonucleotide described herein. In other words, the sense strand comprises at least one nucleotide of formula (II). In some embodiments of any one of the aspects described herein, the antisense strand is an oligonucleotide described herein. In other words, the antisense strand comprises at least one nucleotide of formula (II). Preferably, the sense strand comprises at least one nucleotide of formula (II).

[0313] In some embodiments of the various aspects described herein, the antisense strand is substantially complementary to a target nucleic acid, e.g., a target gene or a target mRNA gene, and the dsRNA is capable of directing targeted cleavage of the target nucleic acid.

[0314] In order for dsRNA molecules to be more effective in vivo, antisense strand must have some metabolic stability.In other words, in order for dsRNA molecules to be more effective in vivo, after a certain period of time after administration, some amount of antisense strand may need to be present in vivo.Therefore, in some embodiments, after in vivo administration on the 5th day, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example, in mouse liver.In some embodiments, after in vivo administration on the 6th day, at least 40%, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example, in mouse liver. In some embodiments, after in vivo administration on the 7th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver.In some embodiments, after in vivo administration on the 8th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver.In some embodiments, after in vivo administration on the 9th day, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80% of the antisense strand of dsRNA is present in vivo, for example in mouse liver. In some embodiments, 10 days after in vivo administration, at least 40%, e.g., at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, e.g., in the mouse liver.In some embodiments, after in vivo administration on the 11th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration on the 12th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration on the 13th day, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%. In some embodiments, after in vivo administration, 14 days later, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.In some embodiments, after in vivo administration, 15 days later, at least 40% of the antisense strand of dsRNA exists in vivo, for example, in mouse liver, for example, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% or at least 80%.

[0315] chain length Various aspects of the present disclosure include double-stranded nucleic acids, e.g., dsRNAs comprising an antisense strand and a sense strand. Note that each strand can range from 12 to 40 nucleotides in length. For example, each strand can independently be 14 to 40 nucleotides, 17 to 37 nucleotides, 25 to 37 nucleotides, 27 to 35 nucleotides, 17 to 23 nucleotides, 17 to 21 nucleotides, 17 to 19 nucleotides, 19 to 25 nucleotides, 19 to 23 nucleotides, 19 to 21 nucleotides, 21 to 25 nucleotides, 21 to 23 nucleotides, 25 to 35 nucleotides, 26 to 35 nucleotides, 27 to 34 nucleotides, 28 to 32 nucleotides, or 29 to 31 nucleotides in length. Without limitation, the sense and antisense strands can be equal or unequal in length. In some embodiments, the antisense strand is longer than the sense strand, e.g., by 1, 2, 3, 4, or 5 nucleotides.

[0316] 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 some embodiments, the antisense strand is 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32 nucleotides in length. In some embodiments, the antisense strand is 21, 22, 23, 24, or 25 nucleotides in length. In some preferred embodiments, the antisense strand is 22, 23, or 25 nucleotides in length.

[0317] Like the antisense strand, the sense strand can be 18 to 35 nucleotides in length in some embodiments. In some embodiments, the sense strand is 21 to 25, 19 to 25, 19 to 21, or 21 to 23 nucleotides in length. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 nucleotides in length. In some embodiments, the antisense strand is 19, 21, 22, or 23 nucleotides in length. In some preferred embodiments, the sense strand is 21 nucleotides in length.

[0318] In some embodiments of any one of the aspects described herein, the sense strand is 15 nucleotides in length and the antisense strand is 18, 19, 20, 21, or 22 (e.g., 20) nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 19 nucleotides in length and the antisense strand is 19, 20, or 21 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20 nucleotides in length and the antisense strand is 20, 21, or 22 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 21 nucleotides in length and the antisense strand is 21, 22, or 23 nucleotides in length. In some embodiments of any one of the aspects described herein, the sense strand is 20-24 (e.g., 22) nucleotides in length and the antisense strand is 34-38 (e.g., 36) nucleotides in length.

[0319] In some embodiments, the antisense strand is 21, 22, or 25 nucleotides in length and the sense strand is 21 nucleotides in length.

[0320] double stranded region The sense strand and the antisense strand typically form a double-stranded region. Generally, the double-stranded region (duplex region) is 12 to 40 nucleotide base pairs in length, 15 to 35 nucleotide base pairs in length, 17 to 30 nucleotide base pairs in length, 25 to 35 nucleotide base pairs in length, 27 to 35 nucleotide base pairs in length, 17 to 23 nucleotide base pairs in length, 17 to 21 nucleotide base pairs in length, 17 to 19 nucleotide base pairs in length, 19 to 25 nucleotide base pairs in length, 19 to 23 nucleotide base pairs in length, 19 to 21 nucleotide base pairs in length, 21 to 25 nucleotide base pairs in length, or 21 to 23 nucleotide base pairs in length. For example, dsRNA has a double-stranded region of 15 to 35 nucleotide pairs in length. In some embodiments, dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 nucleotide base pairs in length.In some embodiments, dsRNA has a duplex region of 19, 20, 21, 22 or 23 nucleotide base pairs in length.In some preferred embodiments, dsRNA has a duplex region of 21 nucleotide base pairs in length.

[0321] overhang In some embodiments, the dsRNA comprises one or more strand overhang regions (i.e., single-stranded regions) and / or capping groups at the 3' or 5' end or both ends of the strand. Without limitation, the overhangs can be 1 to 10 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 1 to 4 nucleotides, 1 to 3 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 2 to 4 nucleotides, 2 to 3 nucleotides, or 1 to 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 from each other. The overhangs can form a mismatch with the target sequence, or can be complementary to the target sequence, or can be other sequences. The first and second strands can also be joined by additional bases, for example, to form a hairpin, or by other non-basic linkers. Without limitation, the overhangs can be present at the 3' end of the sense strand, the antisense strand, or both strands.

[0322] In some embodiments, dsRNA comprises a single overhang.For example, dsRNA has a single overhang, and the overhang is at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides long.In some embodiments, the overhang is at the 3' end of antisense strand.In some particular embodiments, dsRNA comprises a 2-nucleotide overhang at the 3' end of antisense strand.

[0323] dsRNA can also have blunt ends.For example, one end of dsRNA is blunt, and the other end has an overhang.Without being limited, blunt end can be located at the 5' end of antisense strand (or the 3' end of sense strand), or vice versa.Generally, the antisense strand of dsRNA has a nucleotide overhang at the 3' end, and the 5' end is blunt.In some embodiments, dsRNA has a 2-nucleotide overhang at the 3' end of antisense strand, and the 5' end of antisense strand is blunt.

[0324] In some other embodiments, the dsRNA has two blunt ends, ie, at both ends of the dsRNA.

[0325] The nucleotides in the overhang region can each independently be modified or unmodified nucleotides, for example, but not limited to, 2'-sugar modified nucleotides such as 2'-fluoro, 2'-O-methyl, thymidine (T), 2'-O-methoxyethyl-5-methyluridine, 2'-O-methoxyethyl adenosine, 2'-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h'GNA, and any combination thereof. For example, TT (or UU) can be the overhang sequence at either end of either strand. The 5'-overhang or 3'-overhang of the sense strand, antisense strand, or both strands can be phosphorylated. In some embodiments, the overhang region contains two nucleotides with a phosphorothioate internucleotide linkage between them, wherein the two nucleotides in the overhang region can be the same or different.

[0326] The internucleoside linkages in the overhang region can be modified or unmodified internucleotide linkages, for example, the overhang region can include one, e.g., two or more, phosphorothioate internucleoside linkages.

[0327] Nucleic acid modification In some embodiments of any one of the above aspects, the oligonucleotide or double-stranded nucleic acid described herein can contain one or more nucleic acid modifications. For example, the oligonucleotide or double-stranded nucleic acid described herein can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleic acid modifications. It should be noted that when two or more modifications are present, they can be the same, different, or some combination of the same and different modifications. Furthermore, all modifications can be present on one strand of the double-stranded nucleic acid. In some embodiments, both strands of the double-stranded nucleic acid contain at least one nucleic acid modification. When both strands contain at least one modification, the modifications can be the same, different, or some combination of the same and different modifications.

[0328] 2'-Fluoro-modified nucleotides In some embodiments, oligonucleotide or dsRNA described herein can further comprise 2'-fluoro nucleotide, i.e., 2'-fluoro modification.For example, oligonucleotide or dsRNA described herein can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more 2'-fluoro nucleotide.Please note that all 2'-fluoro nucleotides can be present in one strand of dsRNA.

[0329] The antisense strand can contain at least one or more 2'-fluoro nucleotides. For example, the antisense strand can contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) additional 2'-fluoro nucleotides. In some embodiments, the antisense strand contains 1, 2, 3, 4, 5, or 6 2'-fluoro nucleotides. Without limitation, the additional 2'-fluoro modification(s) in the antisense strand can be located at any position. In some embodiments, the antisense strand contains at least three 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at positions 2, 14, and 16 from the 5' end. In other embodiments, the antisense strand contains at least four 2'-fluoro nucleotides. For example, the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand contains at least five 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 9, 14 and 16 from the 5' end. In some further embodiments, the antisense strand comprises at least six 2'-fluoro nucleotides. For example, the antisense strand comprises 2'-fluoro nucleotides at least at positions 2, 6, 8, 9, 14 and 16 from the 5' end.

[0330] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be the nucleotide at the 5'-end or 3'-end of destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5'-end and 3'-end of destabilizing modification, i.e., at the -1 and +1 positions from the position of destabilizing modification.In some embodiments, antisense strand comprises at least two 2'-fluoro nucleotides at the 3'-end of destabilizing modification, i.e., at the +1 and +2 positions from the position of destabilizing modification.

[0331] In some embodiments, the antisense strand does not contain 2'-fluoro nucleotides at positions 3-9 counting from the 5' end.

[0332] The sense strand can contain at least one or more 2'-fluoro nucleotides. For example, the antisense strand can contain at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) 2'-fluoro nucleotides. In some embodiments, the sense strand contains 1, 2, 3, 4, or 5 2'-fluoro nucleotides. For example, the sense strand contains three or four 2'-fluoro nucleotides. Without limitation, the 2'-fluoro modification in the sense strand can be present at any position. In some embodiments, the sense strand contains at least three 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, and 11th positions from the 5' end, or the 11th, 13th, and 15th positions from the 3' end. In other embodiments, the sense strand contains at least four 2'-fluoro nucleotides. For example, the sense strand contains 2'-fluoro nucleotides at least at the 7th, 9th, 10th, and 11th positions from the 5' end, or the 11th, 12th, 13th, and 15th positions from the 3' end. In some embodiments of any one of the aspects described herein, the sense strand comprises 2'-fluoro nucleotides at positions 9, 10, and 11, counting from the 5' end of the sense strand, or at positions 11, 12, and 13, counting from the 3' end of the sense strand. In some embodiments, the sense strand comprises blocks of 2, 3, or 4 clustered 2'-fluoro nucleotides.

[0333] In some embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to the 11th, 12th, and 15th positions of the antisense strand, counting from the 5' end of the antisense strand. In other embodiments, the sense strand comprises 2'-fluoro nucleotides at positions opposite or complementary to the 11th, 12th, 13th, and 15th positions of the antisense strand, counting from the 5' end of the antisense strand.

[0334] In some embodiments, the sense strand comprises blocks of 2, 3, or 4 clustered 2'-fluoro nucleotides.

[0335] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification in the antisense strand of the duplex.

[0336] In some embodiments, both sense strand and antisense strand comprise at least one, for example, at least two, 2'-fluoro nucleotides.2'-fluoro modification can be present on any nucleotide of sense strand or antisense strand.For example, 2'-fluoro modification can be present on all nucleotides on sense strand and / or antisense strand, or each 2'-fluoro modification can be present on sense strand or antisense strand in an alternating pattern, or both sense strand and antisense strand comprise 2'-fluoro modification in an alternating pattern.The alternating pattern of 2'-fluoro modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of 2'-fluoro modification on sense strand can be shifted relative to the alternating pattern of 2'-fluoro modification on antisense strand.

[0337] 2'-deoxy (2'-H, DNA) nucleotides In some embodiments, the oligonucleotide or dsRNA described herein can further comprise 2'-deoxy (for example, 2'-H or DNA) nucleotide.For example, the oligonucleotide or dsRNA described herein can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more DNA nucleotides.Please note that all DNA nucleotides can be present in one strand of dsRNA in dsRNA.

[0338] In some embodiments, the antisense strand can contain at least one (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) DNA nucleotide. In some embodiments, the antisense strand can contain 2, 3, 4, 5, or 6 DNA nucleotides. Without limitation, the DNA nucleotides in the antisense strand can be located at any position. For example, the antisense strand can contain 2'-deoxynucleotides at 1, 2, 3, 4, 5, or 6 of the 2nd, 5th, 7th, 12th, 14th, and 16th positions, counting from the 5'-end of the antisense strand. In a non-limiting example, the antisense strand can contain 2'-deoxynucleotides at 1, 2, 3, or 4 of the 2nd, 5th, 7th, and 12th positions, counting from the 5'-end of the antisense strand.

[0339] In some embodiments, the antisense strand comprises a 2'-deoxynucleotide at the 2nd or 12th position counting from the 5' end of the antisense strand. For example, the antisense strand comprises a 2'-deoxynucleotide at the 12th position counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a 2'-deoxynucleotide at the 5th and 7th positions counting from the 5' end of the antisense strand. For example, the antisense strand comprises a 2'-deoxynucleotide at the 5th, 7th, and 12th positions counting from the 5' end of the antisense strand. In some embodiments, the antisense strand comprises a 2'-deoxynucleotide at the 2nd, 5th, and 7th positions counting from the 5' end of the antisense strand. In other embodiments, the antisense strand comprises at least four DNA nucleotides. For example, the antisense strand comprises at least DNA nucleotides at the 2nd, 5th, 7th, and 12th positions counting from the 5' end. In some further embodiments, the antisense strand comprises at least five DNA nucleotides. In still some further embodiments, the antisense strand comprises at least six DNA nucleotides. For example, the antisense strand contains DNA nucleotides at positions 2, 5, 7, 12, 14, and 16 counting from the 5' end.

[0340] In some embodiments of any one of the aspects described herein, the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, and a 2'-fluoro nucleotide at position 14 of the antisense strand.

[0341] As described herein, the dsRNA can include at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications in the central region of the sense and / or antisense strand. For example, at least one of the sense strand and the antisense strand can include at least one, e.g., at least two, at least three, at least four, at least five, at least six, at least seven, or more 2'-deoxy modifications at positions 5-17, e.g., 6-16, 6-15, 6-14, 6-13, 6-12, 7-15, 7-14, 7-13, 7-12, 8-16, 8-15, 8-14, 8-13, 8-12, 9-16, 9-15, 9-14, 9-13, 9-12, 10-16, 10-15, 10-14, 10-13, or 10-12, counting from the 5' end of the sense strand or antisense strand.

[0342] In some embodiments, both sense strand and antisense strand contain at least one DNA nucleotide.The DNA nucleotide can be present at any nucleotide of sense strand or antisense strand.For example, the DNA nucleotide can be present at every nucleotide of sense strand and / or antisense strand, or each DNA nucleotide can be present in an alternating pattern on sense strand or antisense strand, or both sense strand and antisense strand contain DNA nucleotide in an alternating pattern.The alternating pattern of the DNA nucleotide on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of the DNA nucleotide on the sense strand can be shifted relative to the alternating pattern of the DNA nucleotide on the antisense strand.

[0343] In some embodiments, the dsRNA contains at least three 2'-deoxy modifications, which are located at positions 2 and 14 of the antisense strand, counting from the 5' end of the antisense strand, and at position 11 of the sense strand, counting from the 5' end of the sense strand.

[0344] In some embodiments, the dsRNA contains at least five 2'-deoxy modifications, the 2'-deoxy modifications being located at positions 2, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0345] In some embodiments, the dsRNA contains at least seven 2'-deoxy modifications, the 2'-deoxy modifications being at positions 2, 5, 7, 12, and 14 of the antisense strand, counting from the 5' end of the antisense strand, and positions 9 and 11 of the sense strand, counting from the 5' end of the sense strand.

[0346] In one non-limiting example, the sense strand does not contain a 2'-deoxynucleotide at position 11, counting from the 5' end of the sense strand.

[0347] 2'-OMe nucleotides In some embodiments, the oligonucleotides or dsRNAs described herein can contain at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more 2'-OMe nucleotides. Note that all of the 2'-OMe nucleotides can be present on one strand of the dsRNA.

[0348] In some embodiments, the antisense strand can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 or more 2'-OMe nucleotides. Without limitation, the 2'-OMe nucleotides in the antisense strand can be located at any position. In some embodiments of any one of the aspects described herein, all remaining nucleotides in the antisense strand are 2'-OMe nucleotides.

[0349] In some embodiments, the antisense strand does not contain 2'-OMe nucleotides at positions 2, 14, and 16 from the 5' end. In some other embodiments, the antisense strand does not contain 2'-OMe nucleotides at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand does not contain 2'-OMe nucleotides at positions 2, 6, 9, 14, and 16 from the 5' end. In still some further embodiments, the antisense strand does not contain 2'-OMe nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0350] The sense strand can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 or more 2'-OMe nucleotides. Without limitation, the 2'-OMe nucleotides in the sense strand can be located at any position. In some embodiments, the remaining nucleotides in the sense strand are all 2'-OMe nucleotides.

[0351] In some embodiments, the sense does not contain 2'-OMe nucleotides at least at positions 7, 10, and 11 from the 5' end or positions 11, 12, and 15 from the 3' end. In other embodiments, the sense does not contain 2'-OMe nucleotides at least at positions 7, 9, 10, and 11 from the 5' end or positions 11, 12, 13, and 15 from the 3' end.

[0352] In some embodiments, both sense strand and antisense strand comprise at least one 2'-OMe nucleotide.2'-OMe modification can be present on any nucleotide of sense strand or antisense strand.For example, 2'-OMe modification can be present on all nucleotides of sense strand and / or antisense strand, or each thermostabilizing modification can be present on sense strand or antisense strand in an alternating pattern, or both sense strand and antisense strand comprise 2'-OMe modification in an alternating pattern.The alternating pattern of thermostabilizing modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of thermostabilizing modification on sense strand can be shifted relative to the alternating pattern of 2'-OMe modification on antisense strand.

[0353] Other modified nucleotides In some embodiments, oligonucleotide or dsRNA described herein can comprise locked nucleic acid (LNA).For example, oligonucleotide or dsRNA described herein 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 or 20 or more LNA modifications.Please note that all LNA nucleotides can be present in one strand of dsRNA.

[0354] In some embodiments, both sense strand and antisense strand comprise at least LNA modification.LNA modification can be present on any nucleotide of sense strand or antisense strand.For example, LNA modification can be present on all nucleotides of sense strand and / or antisense strand, or each LNA modification can be present on sense strand or antisense strand in alternating pattern, or both sense strand and antisense strand comprise LNA modification in alternating pattern.The alternating pattern of LNA modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of LNA modification on sense strand can be shifted relative to the alternating pattern of 2'-fluoro modification on antisense strand.

[0355] The antisense strand can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications. Without being limited thereto, the LNA modifications in the antisense strand can be present in any position.

[0356] The sense strand can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications. Without being limited thereto, the LNA modifications in the sense strand can be present at any position. In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more LNA modifications, and the antisense strand does not comprise 2'-fluoro nucleotides at positions 3 to 9 counting from the 5' end.

[0357] The oligonucleotide or dsRNA described herein can comprise a bridged nucleic acid (BNA). For example, the oligonucleotide or dsRNA described herein 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, or 20 or more BNA modifications. Without being limited thereto, all BNA nucleotides can be present in one dsRNA. In some embodiments, both the sense strand and the antisense strand contain at least a BNA modification. The BNA modification can be present on any nucleotide of the sense strand or the antisense strand. For example, the BNA modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each BNA modification can be present in an alternating pattern on the sense strand or the antisense strand, or both the sense strand and the antisense strand contain the BNA modification in an alternating pattern. The alternating pattern of BNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of BNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0358] The antisense strand can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more BNA modifications. Without being limited thereto, the BNA modifications in the antisense strand can be present in any position.

[0359] The sense strand can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more BNA modifications. Without being limited thereto, the BNA modifications in the sense strand can be present at any position. In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more BNA modifications, and the antisense strand does not comprise 2'-fluoro nucleotides at positions 3 to 9 counting from the 5' end.

[0360] The oligonucleotide or dsRNA described herein can contain cyclohexene nucleic acid (CeNA). For example, the oligonucleotide or dsRNA described herein can contain at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more CeNA modifications. Without being limited thereto, all CeNA nucleotides can be present on one strand of dsRNA. In some embodiments, both the sense strand and the antisense strand contain CeNA modifications. CeNA modifications can be present on any nucleotide of the sense strand or antisense strand. For example, CeNA modifications can be present on every nucleotide on the sense strand and / or antisense strand, or each CeNA modification can be present in an alternating pattern on the sense strand or antisense strand, or both the sense strand and the antisense strand contain CeNA modifications in an alternating pattern. The alternating pattern of CeNA modifications on the sense strand can be the same as or different from the antisense strand, and the alternating pattern of CeNA modifications on the sense strand can be shifted relative to the alternating pattern of 2'-fluoro modifications on the antisense strand.

[0361] The antisense strand can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more CeNA modifications. Without being limited thereto, the CeNA modifications in the antisense strand can be present in any position.

[0362] The sense strand can comprise at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more CeNA modifications. Without being limited thereto, the CeNA modifications in the sense strand can be present at any position. In some embodiments, the sense strand comprises at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more CeNA modifications, and the antisense strand does not comprise 2'-fluoro nucleotides at positions 3 to 9 counting from the 5' end.

[0363] The oligonucleotide or dsRNA described herein can comprise thermostabilizing modification.For example, the oligonucleotide or dsRNA described herein can comprise at least four, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 or more thermostabilizing modifications.All thermostabilizing modifications can be present in one strand of dsRNA.

[0364] In some embodiments, both the sense strand and the antisense strand contain at least one, for example, two, three, four or more thermostabilizing modifications.The thermostabilizing modification can be present on any nucleotide of the sense strand or the antisense strand.For example, the thermostabilizing modification can be present on every nucleotide of the sense strand and / or the antisense strand, or each thermostabilizing modification can be present on the sense strand or the antisense strand in an alternating pattern, or both the sense strand and the antisense strand contain thermostabilizing modifications in an alternating pattern.The alternating pattern of the thermostabilizing modification on the sense strand can be the same or different from that of the antisense strand, and the alternating pattern of the thermostabilizing modification on the sense strand can be shifted relative to the alternating pattern of the thermostabilizing modification on the antisense strand.

[0365] The antisense strand can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more thermostabilizing modifications. In some embodiments, the antisense strand contains 2, 3, 4, 5, or 6 thermostabilizing modifications. Without limitation, the thermostabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand contains at least three thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at positions 2, 14, and 16 from the 5' end. In other embodiments, the antisense strand contains at least four thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at positions 2, 6, 14, and 16 from the 5' end. In some further embodiments, the antisense strand contains at least five thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at positions 2, 6, 9, 14, and 16 from the 5' end. In still some further embodiments, the antisense strand contains at least six thermostabilizing modifications. For example, the antisense strand contains thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0366] The sense strand can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more thermostabilizing modifications. In some embodiments, the antisense strand contains 2, 3, 4, or 5 thermostabilizing modifications. For example, the sense strand contains 3 or 4 thermostabilizing modifications. Without limitation, the thermostabilizing modifications in the sense strand can be located at any position. In some embodiments, the sense strand contains at least three thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at least at the 7th, 10th, and 11th positions from the 5' end. In other embodiments, the sense strand contains at least four thermostabilizing modifications. For example, the sense strand contains thermostabilizing modifications at least at the 7th, 9th, 10th, and 11th positions from the 5' end.

[0367] In some embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In other embodiments, the sense strand comprises thermostabilizing modifications at positions opposite or complementary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5' end of the antisense strand. In some embodiments, the sense strand comprises blocks of 2, 3, or 4 clustered thermostabilizing modifications.

[0368] In some embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0369] In some embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 14, and 16 from the 5' end. In some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 9, 14, and 16 from the 5' end. In yet some other embodiments, the sense strand comprises thermostabilizing modifications at least at positions 7, 9, 10, and 11 from the 5' end, and the antisense strand comprises thermostabilizing modifications at least at positions 2, 6, 8, 9, 14, and 16 from the 5' end.

[0370] In some embodiments, the sense strand does not contain a thermostabilizing modification at a position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0371] Exemplary thermostabilizing modifications include, but are not limited to, 2'-fluoro modifications and locked nucleic acids (LNAs).

[0372] Internucleoside linkage As used herein, "internucleoside linkage" refers to a covalent linkage between adjacent nucleosides. Two major classes of internucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing linkages include, but are not limited to, phosphodiester (P=O), phosphotriester, methylphosphonate, phosphoramidate, and phosphorothioate (P=S). Representative non-phosphorus-containing linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-OC(O)-S-), thionocarbamate (-OC(O)(NH)-S-), siloxane (-O-Si(H)2-O-), and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages can be used to alter, typically increase, the nuclease resistance of oligonucleotide compounds compared to natural phosphodiester linkages. In certain embodiments, linkages having chiral atoms can be prepared as racemic mixtures, as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods for preparing phosphorus(III)-containing and non-phosphorus(III)-containing linkages are well known to those skilled in the art.

[0373] The phosphate group in the internucleoside linkage can be modified by replacing one of its oxygen atoms with a different substituent. One result of this modification can be increased resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphodiester internucleoside linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e., alkyl group, aryl group, etc.), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorus(III) atom in the unmodified phosphate group is achiral. However, if one of the non-bridging oxygens is replaced with one of the atoms or groups of atoms described above, the phosphorus(III) atom becomes chiral. In other words, the phosphorus(III) atom in the phosphate group modified in this manner is an asymmetric center. The asymmetric phosphorus(III) atom can have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp).

[0374] In phosphorodithioates, both non-bridging oxygens are replaced with sulfur. The phosphorus center in phosphorodithioates is achiral, which eliminates the formation of oligonucleotide diastereomers. Therefore, without wishing to be bound by theory, modification of both non-bridging oxygens to remove chiral centers, such as forming phosphorodithioates, may be desirable in that it cannot produce diastereomeric mixtures. The non-bridging oxygens can be independently O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0375] Phosphodiester internucleoside linkages can also be modified by replacement of the bridging oxygen (i.e., the oxygen linking the phosphate to the sugar of the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylenesulfonates). Replacement can be at one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3'-oxygen of the nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5'-oxygen of the nucleoside, replacement with nitrogen is preferred.

[0376] Modified phosphate linkages in which at least one of the oxygens linked to the phosphate is replaced or the phosphate group is replaced with a non-phosphorus(III) group are also referred to as "non-phosphodiester intersugar linkages" or "non-phosphodiester linkers."

[0377] In certain embodiments, the phosphate group can be replaced with a non-phosphorus-containing connector, e.g., a dephospho linker. A dephospho linker is also referred to herein as a non-phosphodiester linker. Without wishing to be bound by theory, because the charged phosphodiester group is the reactive center in nucleolytic degradation, its replacement with a neutral structural mimic should confer enhanced nuclease stability. Again, without wishing to be bound by theory, in some embodiments it may be desirable to introduce a modification in which the charged phosphate group is replaced with a neutral moiety.

[0378] Examples of moieties that can replace the phosphate group include amide (e.g., amide-3 (3'-CH2-C(=O)-N(H)-5') and amide-4 (3'-CH2-N(H)-C(=O)-5')), hydroxylamino, siloxane (dialkylsiloxane), carboxamide, carbonate, carboxymethyl, carbamate, carboxylic acid ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonic acid ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3'-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, amino), methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ether (C3'-O-C5'), thioether (C3'-S-C5'), thioacetamide (C3'-N(H)-C(=O)-CH2-S-C5', C3'-OP(O)-O-SS-C5', C3'-CH2-NH-NH-C5', 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5', as well as non-ionic linkages containing mixed N, O, S and CH2 component parts. For example, "Carbohydrate See "Modifications in Antisense Research," edited by YS Sanghvi and PD Cook, ACS Symposium Series 580, Chapters 3 and 4 (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amide, carbamate, and ethylene oxide linkers.

[0379] It is well known to those skilled in the art that in certain instances, replacement of a non-bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the adjacent 2'-OH, and therefore modification of the non-bridging oxygen may in many instances require modification of the 2'-OH, e.g., a modification that does not participate in cleavage of the adjacent intersugar linkage, e.g., arabinose sugars, 2'-O-alkyl, 2'-F, LNA, and ENA.

[0380] Preferred non-phosphodiester internucleoside linkages include phosphorothioates, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more for the Sp isomer, phosphorothioates in an enantiomeric excess of at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or more for the Rp isomer, phosphorodithioates, phosphotriesters, aminoalkylphosphotrioesters, alkylphosphonates (e.g., methylphosphonates), selenophosphates, phosphoramidates (e.g., N-alkylphosphoramidates), and boranophosphonates.

[0381] Further exemplary non-phosphorus-containing internucleoside linking groups are described in U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,264,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, 5,541,307, 5,561,225, Nos. 5,596,086, 5,602,240, 5,610,289, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, 5,792,608, 5,646,269 and 5,677,439, the contents of each of which are incorporated herein by reference.

[0382] In some embodiments of any one of the above aspects, the antisense strand and / or the sense strand comprise one or more neutral internucleoside linkages that are non-ionic. Suitable neutral internucleoside linkages include phosphotriester, methylphosphonate, MMI (3'-CH2-N(CH3)-O-5'), amide-3 (3'-CH2-C(=O)-N(H)-5'), amide-4 (3'-CH2-N(H)-C(=O)-5'), formacetal (3'-O-CH2-O-5'), and thioformacetal (3'-S-CH2-O-5'); non-ionic linkages containing siloxanes (dialkylsiloxanes), carboxylate esters, carboxamides, sulfides, sulfonate esters, and / or amides (see, e.g., "Carbohydrate Modifications in Antisense Research," edited by Y.S. Sanghvi and P.D. Cook, ACS Symposium Series, Vol. 1, No. 1, pp. 111-114, 2002). 580, Chapters 3 and 4 (pp. 40-65)); and non-ionic linkers containing mixed N, O, S, and CH2 component parts.

[0383] In some embodiments, the non-phosphodiester backbone linkages are selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate, and phosphoramidate backbone linkages.

[0384] In some embodiments of any one of the aspects described herein, the internucleoside linkage is: TIFF2025534336000068.tif25128, where R IL1 and R IL2 is, independently at each occurrence, absent, O, S, CH, NR (where R is hydrogen, alkyl, aryl), or optionally substituted alkylene, where the alkylene backbone can contain one or more of O, S, SS, and NR (where R is hydrogen, alkyl, aryl) internally and / or terminally; R IL3 and R IL4are each independently O, OR (R is hydrogen, alkyl, or aryl), S, Se, BR3 (R is hydrogen, alkyl, or aryl), BH3 - , C (i.e., alkyl, aryl, etc.), H, NR2 (where R is hydrogen, alkyl, aryl), alkyl, or aryl. R IL1 and R IL2 replaces the oxygen linked to the 5' carbon of the first nucleoside sugar, and R IL1 and R IL2 It is understood that the other of replaces the oxygen linked to the 3' (or 2') carbon of the second nucleoside sugar.

[0385] In some embodiments of any one of the above aspects, R IL1 , R IL2 , R IL3 and R IL4 are all O.

[0386] In some embodiments, R IL1 and R IL2 is O and R IL3 and R IL4 At least one of them is other than O. For example, R IL3 and R IL4 one of which is S and the other is O, or R IL3 and R IL4 Both are S.

[0387] In some embodiments of any one of the aspects described herein, R 23 is a bond to a modified internucleoside linkage, such as the structure: TIFF2025534336000069.tif25128 to the internucleoside linkage, where R IL1 , R IL2 , R IL3 and R IL4 At least one of the is not O. For example, R IL3 and R IL4 At least one of them is S.

[0388] In some embodiments of any one of the aspects described herein, R 23 or R 22 is bonded to a phosphorothioate internucleoside linkage, e.g., the structure: TIFF2025534336000070.tif25128 to the internucleoside linkage, where R IL1 and R IL2 At least one of the groups is O, and R IL3 and R IL4 One of the is O and R IL3 and R IL4 The other of these is S.

[0389] In some embodiments of any one of the aspects described herein, R 23 or R 22 is a bond to a phosphodiester internucleoside linkage, e.g., the structure: TIFF2025534336000071.tif25128 to the internucleoside linkage, where R IL1 , R IL2 , R IL3 and R IL4 is O.

[0390] In some embodiments of any one of the above aspects, the antisense strand and / or the sense strand can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more, modified internucleoside linkages. For example, the antisense strand and / or the sense strand can comprise 1, 2, 3, 4, 5, or 6 modified internucleoside linkages. For example, the antisense strand and / or the sense strand comprises 1, 2, 3, or 4 modified internucleoside linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least two modified internucleoside linkages within the first five nucleotides counting from the 5'-end of the strand and further comprises at least two modified internucleoside linkages within the first five nucleotides counting from the 3'-end of the strand. For example, the antisense strand and / or the sense strand contain modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the strand, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the strand.

[0391] In some embodiments of any one of the above aspects, the modified internucleoside linkage is phosphorothioate. Thus, in some embodiments of any one of the above aspects, the antisense strand and / or the sense strand comprises one or more, for example, 1, 2, 3, 4, 5, 6, 7, or 8 or more, phosphorothioate internucleoside linkages. For example, the antisense strand and / or the sense strand comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. For example, the antisense strand and / or the sense strand comprises 1, 2, 3, or 4 phosphorothioate internucleoside linkages. In some embodiments, the antisense strand and / or the sense strand comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the strand and further comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 3' end of the strand. For example, the antisense strand and / or the sense strand contain modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the strand, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the strand.

[0392] phosphorothioate The oligonucleotides or dsRNAs described herein can contain at least one, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more phosphorothioate or methylphosphonate internucleotide linkages. The phosphorothioate or methylphosphonate internucleotide linkage modifications can be present on any nucleotide of the oligonucleotides or dsRNAs described herein.

[0393] In dsRNA, phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage modification can be present at any position of the chain, on any nucleotide of sense strand or antisense strand or both.For example, internucleotide linkage modification can be present at all nucleotides on sense strand and / or antisense strand, or each internucleotide linkage modification can be present on sense strand or antisense strand in alternating pattern, or sense strand or antisense strand contains both internucleotide linkage modifications in alternating pattern.The alternating pattern of internucleotide linkage modification on sense strand can be the same or different from that of antisense strand, and the alternating pattern of internucleotide linkage modification on sense strand can be shifted relative to the alternating pattern of internucleotide linkage modification on antisense strand.

[0394] In some embodiments, dsRNA comprises phosphorothioate internucleotide linkage modification or methylphosphonate internucleotide linkage modification in overhang region.For example, overhang region comprises two nucleotides with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage between two nucleotides.Internucleotide linkage modification can be used to connect overhang nucleotide with the paired nucleotide at the end in double-stranded region.For example, at least 2, 3 or 4 or all overhang nucleotides can be linked with phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage, and optionally there can be an additional phosphorothioate internucleotide linkage or methylphosphonate internucleotide linkage that connects the overhang nucleotide with the paired nucleotide adjacent to the overhang nucleotide.For example, there can be at least two phosphorothioate internucleotide linkages between the three nucleotides at the end, and two of these three nucleotides can be overhang nucleotides, and the third can be the paired nucleotide adjacent to the overhang nucleotide. Preferably, these three terminal nucleotides can be at the 3' end of the antisense strand.

[0395] In some embodiments, the sense strand comprises 1 to 10 blocks of 2 to 10 clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the sense strand, and the sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0396] In some embodiments, the antisense strand comprises two blocks of two clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either a phosphorothioate linkage or a methylphosphonate linkage or a phosphate linkage.

[0397] In some embodiments, the antisense strand comprises two blocks of three clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0398] In some embodiments, the antisense strand comprises two blocks of four clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0399] In some embodiments, the antisense strand comprises two blocks of five clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0400] In some embodiments, the antisense strand comprises two blocks of six clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0401] In some embodiments, the antisense strand comprises two blocks of seven clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0402] In some embodiments, the antisense strand comprises two blocks of eight clustered phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0403] In some embodiments, the antisense strand comprises two blocks of nine clustered phosphorothioate or methylphosphonate internucleotide linkages separated by one, two, three, or four phosphate internucleotide linkages, one of the phosphorothioate or methylphosphonate internucleotide linkages being located at any position in the antisense strand, and the antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate, and phosphate internucleotide linkages, or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkages.

[0404] In some embodiments, the dsRNA comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides at one or both ends of the sense and / or antisense strands can be linked by phosphorothioate or methylphosphonate internucleotide linkages.

[0405] In some embodiments, the dsRNA comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 internal regions of the duplex of each of the sense and / or antisense strands. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in positions 8 to 16 of the duplex region, counting from the 5' end of the sense strand, can be linked by phosphorothioate-methylphosphonate internucleotide linkages, and the dsRNA can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modifications within 1 to 10 of the terminal positions.

[0406] In some embodiments, the dsRNA comprises 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 5 (counting from the 5' end) of the sense strand and 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1 to 5 (counting from the 3' end) of the sense strand, and 1 to 5 phosphorothioate or methylphosphonate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand and 1 to 5 within positions 1 to 5 (counting from the 3' end).

[0407] In some embodiments, the dsRNA comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate or methylphosphonate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0408] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 18-23 (counting from the 3' end) of the antisense strand.

[0409] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0410] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand.

[0411] In some embodiments, the dsRNA comprises one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0412] In some embodiments, the dsRNA contains one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) and one within positions 1-5 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand.

[0413] In some embodiments, the dsRNA contains one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 5' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand.

[0414] In some embodiments, the dsRNA contains two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0415] In some embodiments, the dsRNA contains two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one within positions 1-5 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand.

[0416] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0417] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification within positions 1-5 (counting from the 3' end) of the antisense strand, and one phosphorothioate internucleotide linkage modification within positions 1-2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications within positions 1-5 (counting from the 3' end) of the antisense strand.

[0418] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 (counting from the 3' end) of the antisense strand.

[0419] In some embodiments, the dsRNA comprises one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end) of the antisense strand.

[0420] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 3' end) of the antisense strand.

[0421] In some embodiments, the dsRNA comprises one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) of the antisense strand.

[0422] In some embodiments, the dsRNA comprises two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end) of the sense strand, and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 3' end) of the antisense strand.

[0423] In some embodiments, the dsRNA has one phosphorothioate internucleotide linkage modification at position 1 (counting from the 5' end) and one phosphorothioate internucleotide linkage modification at position 1 (counting from the 3' end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 5' end) and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 (counting from the 3' end) of the antisense strand.

[0424] In some embodiments, the sense strand can include 0, 1, 2, 3, or 4 phosphorothioate internucleotide linkages, for example, the sense strand includes a phosphorothioate internucleotide linkage between the first and second nucleotide and between the second and third nucleotide (counting from the 5' end).

[0425] In some embodiments, the antisense strand can contain 1, 2, 3, or 4 phosphorothioate internucleotide linkages. For example, the sense strand contains phosphorothioate internucleotide linkages between the first and second nucleotides (counting from the 3' end) and between the second and third nucleotides. In a further example, the antisense strand contains phosphorothioate internucleotide linkages between the first and second nucleotides (counting from the 5' end), between the second and third nucleotides (counting from the 5' end), between the first and second nucleotides (counting from the 3' end), and between the second and third nucleotides (counting from the 3' end).

[0426] In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between the first and second nucleotides (counting from the 5' end) and between the second and third nucleotides (counting from the 5' end), and the antisense strand comprises a phosphorothioate internucleotide linkage between the first and second nucleotides (counting from the 3' end) and between the second and third nucleotides (counting from the 5' end). For example, the sense strand comprises a phosphorothioate internucleotide linkage between the first and second nucleotides (counting from the 5' end) and between the second and third nucleotides (counting from the 5' end), and the antisense strand comprises a phosphorothioate internucleotide linkage between the first and second nucleotides (counting from the 5' end), between the second and third nucleotides (counting from the 5' end), between the first and second nucleotides of the oligo (counting from the 3' end), and between the second and third nucleotides (counting from the 5' end).

[0427] 5'-modified In some embodiments, the dsRNA can be 5' phosphorylated or contain a phosphoryl analog at the 5' end of the antisense strand and / or the sense strand. For example, the antisense strand can be 5' phosphorylated or contain a phosphoryl analog at the 5' end. Exemplary 5'-phosphate modifications include those that are compatible with RISC-mediated gene silencing. Suitable modifications include 5'-monophosphate ((HO)2(O)PO-5'), 5'-diphosphate ((HO)2(O)POP(HO)(O)-O-5'), 5'-triphosphate ((HO)2(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-guanosine cap (7-methylated or unmethylated) (7m-GO-5'-(HO)(O)PO-(HO)(O)POP (HO)(O)-O-5'), 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (NO-5'-(HO)(O)PO-(HO)(O)POP(HO)(O)-O-5'), 5'-monothiophosphate (phosphorothioate, (HO)2(S)PO-5'), 5'-monodithiophosphate (phosphorodithioate, (HO)(HS)(S)PO-5'), '), 5'-phosphorothiolates ((HO)2(O)PS-5'), any further combination of oxygen / sulfur substituted monophosphates, diphosphates and triphosphates (e.g., 5'-alpha-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)PO-5'), 5'-alkylphosphonates (R = alkyl = methyl, ethyl, isopropyl, propyl, etc., e.g., RP(OH)(O)-O-5'-, 5'-alkenylphosphonates (i.e., vinyl, substituted vinyl), (OH)2(O)P-5'-CH2-), 5'-alkyl ether phosphonates (R = alkyl ether = methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g., RP(OH)(O)-O-5'-).

[0428] In some embodiments, antisense strand comprises 5'-vinyl phosphonate nucleotide at 5'-end.For example, antisense strand comprises 5'-E-vinyl phosphate nucleotide at 5'-end.In some embodiments, antisense strand comprises 5'-E-vinyl phosphate and the nucleoside at N-1 position, which reduces or inhibits the activity of siRNA compared with the siRNA that has the same antisense strand sequence but is not modified at N-1 position.

[0429] In some embodiments, the sense strand comprises a 5'-morpholino, 5'-dimethylamino, 5'-deoxy, inverted abasic, or inverted abasic locked nucleic acid modification at its 5'-end. In some embodiments, the sense strand comprises a nucleotide of Formula (II) at its 5'-end.

[0430] In some embodiments, the antisense strand comprises a nucleotide of the formula (II-VP) or (II-VP') at its 5' end.

[0431] thermal stability Generally, dsRNA has a melting temperature in the range of about 40°C to about 80°C. For example, dsRNA has a melting temperature with a lower limit in the range of about 40°C, 45°C, 50°C, 55°C, 60°C, or 65°C and an upper limit in the range of about 70°C, 75°C, or 80°C. In some embodiments, dsRNA has 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, dsRNA has a melting temperature in the range of about 57°C to about 67°C. In some specific embodiments, dsRNA has a melting temperature in the range of about 60°C to about 67°C. In some further embodiments, dsRNA has a melting temperature in the range of about 62°C to about 66°C.

[0432] Without wishing to be bound by theory, thermodestabilizing modifications in the seed region of the antisense strand (i.e., 2-9 nucleotides from the 5' end or 23-30 nucleotides from the 3' end of the antisense strand) can reduce or inhibit off-target gene silencing. Thus, the oligonucleotides or dsRNAs described herein can contain at least one (e.g., 1, 2, 3, 4, or 5 or more) thermodestabilizing modifications. In some embodiments, the antisense strand contains at least one (e.g., 1, 2, 3, 4, or 5 or more) thermodestabilizing modifications of the duplex within the first 9 nucleotide positions of the 5' end of the antisense strand or at the 23-31 nucleotide positions from the 3' end.

[0433] The term "thermally destabilizing modification" encompasses modifications that result in a dsRNA with a reduced overall melting temperature (Tm), preferably a Tm that is 1, 2, 3, or 4 degrees lower than the Tm of a dsRNA lacking such modification.

[0434] In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8, or 9 from the 5' end of the antisense strand, or preferably at position 4, 5, 6, 7, or 8. In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, or 9 from the 5' end of the antisense strand. In some other embodiments, the thermally destabilizing modification is located at position 6, 7, or 8 from the 5' end of the antisense strand. In some specific embodiments, the thermally destabilizing modification is located at position 7 from the 5' end of the antisense strand.

[0435] Thermally destabilizing modifications can include, but are not limited to, abasic nucleosides, mismatches with opposing nucleotides in opposing strands, and nucleosides with modified sugars, such as 2'-deoxynucleosides or acyclic nucleosides, such as unlocked nucleic acids (UNAs) or glycol nucleic acids (GNAs).

[0436] Exemplary abasic modifications include, but are not limited to, the following modifications: TIFF2025534336000072.tif67143In the formula, R is H, Me, Et or OMe, R' is H, Me, Et or OMe, R'' is H, Me, Et or OMe, and * represents either R, S or racemic.

[0437] Exemplary destabilizing sugar modifications include, but are not limited to, the following modifications: TIFF2025534336000073.tif159154In the formula, B is a modified or unmodified nucleobase.

[0438] Further sugar modifications include, but are not limited to, the following modifications: TIFF2025534336000074.tif100168In the formula, B is a modified or unmodified nucleobase.

[0439] In some embodiments, the thermodestabilizing modification is TIFF2025534336000075.tif71128; wherein B is a modified or unmodified nucleobase, and the asterisk on each structure represents either R, S, or racemic.

[0440] The term "acyclic nucleotide" refers to any nucleotide having an acyclic ribose sugar, for example, one in which any of the bonds between the ribose carbons (e.g., C1'-C2', C2'-C3', C3'-C4', C4'-O4', or C1'-O4') is absent, and / or at least one of the ribose carbons or oxygens (e.g., C1', C2', C3', C4', or O4'), independently or in combination, is missing from the nucleotide. In some embodiments, an acyclic nucleotide is TIFF2025534336000076.tif35149, wherein B is a modified or unmodified nucleobase, and R 1 and R 2 are independently H, halogen 、OR3, or alkyl, where R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar. The term "UNA" refers to an unlocked acyclic nucleic acid in which one of the sugar bonds has been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses a monomer in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar has been removed (see Mikhailov et al., Tetrahedron Letters, 26(17):2059 (1985) and Fluiter et al., Mol. Biosyst., 10:1039 (2009), each of which is incorporated herein by reference in its entirety). Acyclic derivatives increase backbone flexibility without affecting Watson-Crick pairing. Acyclic nucleotides can be linked by 2'-5' or 3'-5' linkages.

[0441] The term "GNA" refers to glycol nucleic acid, a polymer that is similar to DNA or RNA but differs in that its "backbone" composition consists of repeating glycerol units linked by phosphodiester bonds: Points to TIFF2025534336000077.tif57128.

[0442] The thermally destabilizing modification of the duplex can be a mismatch (i.e., non-complementary base pair) between the thermally destabilizing nucleotide in the double-stranded region of dsRNA and the opposite nucleotide in the opposite strand.Exemplary mismatch 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 mismatch base pairings known in the art can also be applied to the present invention.Mismatches can occur between nucleotides that are either natural nucleotides or modified nucleotides.That is, mismatch base pairing can occur between the nucleobases of each nucleotide, regardless of the modification on the ribose sugar of the nucleotide.In certain embodiments, the dsRNA comprises at least one nucleobase that is a 2'-deoxynucleobase in mismatch pairing, for example, the 2'-deoxynucleobase is in the sense strand.

[0443] In some embodiments, the thermodestabilizing modifications in the seed region of the antisense strand include nucleotides that have impaired WCH binding to a complementary base on the target mRNA. Examples of nucleotides that have impaired WCH binding to a complementary base on the target mRNA include, but are not limited to, nucleotides that include nucleobases independently selected from the following nucleobases: TIFF2025534336000078.tif72135.

[0444] Further examples of abasic nucleotide modifications, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are detailed in WO 2011 / 133876, which is incorporated herein by reference in its entirety.

[0445] Thermally destabilizing modifications can also include universal nucleobases that have reduced or eliminated ability to hydrogen bond with opposing bases, and phosphate modifications.

[0446] In some embodiments, thermal destabilizing modification comprises the nucleotide with non-standard base, for example but not limited to, the nucleobase modification that the ability to form hydrogen bond with the base in the opposite strand is impaired or completely lost.These nucleobase modifications have been evaluated for destabilizing the central region of the double-stranded region of dsRNA, as described in WO 2010 / 0011895, and this document is incorporated herein by reference in its entirety.The exemplary nucleobase modifications of this kind are: TIFF2025534336000079.tif63156.

[0447] In some embodiments, the thermally destabilizing modification includes one or more α-nucleotides, e.g. TIFF2025534336000080.tif20146, where R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0448] Examples of phosphate modifications that are known to decrease the thermal stability of double-stranded nucleic acid duplexes compared to natural phosphodiester linkages include, but are not limited to, the following modifications: TIFF2025534336000081.tif30139

[0449] The alkyl R group can be a C1-C6 alkyl. Specific alkyl R groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, pentyl, and hexyl.

[0450] In some embodiments, the thermolabile modification is an unlocked nucleic acid (UNA) or glycol nucleic acid (GNA). For example, thermolabile modifications can include, but are not limited to, the following mUNA and GNA building blocks: TIFF2025534336000082.tif106162

[0451] In some embodiments, the destabilizing modification is selected from the following modifications: TIFF2025534336000083.tif98128

[0452] In some embodiments, the destabilizing modification is selected from the following modifications: TIFF2025534336000084.tif63151

[0453] In some embodiments, the destabilizing modification is selected from the following modifications: TIFF2025534336000085.tif90154

[0454] In some embodiments, the destabilizing modification is selected from the group consisting of GNA-isoC, GNA-isoG, 5'-mUNA, 4'-mUNA, 3'-mUNA and 2'-mUNA.

[0455] In some embodiments, the destabilizing modified mUNA is selected from the group consisting of: TIFF2025534336000086.tif87152R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0456] In some embodiments, the destabilizing modified mUNA is selected from the group consisting of: TIFF2025534336000087.tif52147R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0457] In some embodiments, the destabilizing modified mUNA is selected from the group consisting of: TIFF2025534336000088.tif85157R=H, OMe;F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0458] In some embodiments, the destabilizing modified mUNA is selected from the group consisting of: TIFF2025534336000089.tif92162R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0459] In some embodiments, the destabilizing modified mUNA is selected from the group consisting of: TIFF2025534336000090.tif54155R = H, OH;OMe;Cl, F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;CCH(alkyne), O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; 7-deazapurine, phenoxazine; G-clamp; non-standard mono-, bi-, and tricyclic heterocycles The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0460] In some embodiments, the modified mUNA is selected from the group consisting of: TIFF2025534336000091.tif84156R=H, OMe;F;OH;O-(CH2)2OMe;SMe, NMe2;NH2;Me;O-nPr;O-alkyl;O-alkylamino; R' = H, Me; B = A; C; 5-Me-C; G; I; U; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidine; C2-modified purine; N8-modified purine; phenoxazine; G-clamp; non-standard monocyclic, bicyclic, and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diaminopurine; pseudocytosine; 2-aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurine The stereochemistry is R or S, with any combination of R and S for unspecified chiral centers.

[0461] In some embodiments, the antisense strand comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) stabilizing modifications. Without limitation, the stabilizing modifications in the antisense strand can be located at any position. In some embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end. In other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 6, 14, and 16, counting from the 5' end. In yet other embodiments, the antisense strand comprises stabilizing modifications at positions 2, 14, and 16, counting from the 5' end. In some embodiments, the antisense strand comprises stabilizing modifications at positions 7, 10, and 11, counting from the 5' end. In other embodiments, the antisense strand comprises stabilizing modifications at positions 7, 9, 10, and 11, counting from the 5' end.

[0462] In some embodiments, antisense strand comprises at least one stabilizing modification adjacent to destabilizing modification.For example, stabilizing modification can be the nucleotide at the 5'-end or 3'-end of destabilizing modification, i.e., the nucleotide at the -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises stabilizing modification at the 5'-end and 3'-end of destabilizing modification, i.e., the nucleotide at the -1 and +1 position from the position of destabilizing modification.

[0463] In some embodiments, the antisense strand comprises at least two stabilizing modifications 3' to the destabilizing modification, i.e., at least two stabilizing modifications at positions +1 and +2 from the position of the destabilizing modification.

[0464] In some embodiments, the sense strand does not contain a thermostabilizing modification at a position opposite or complementary to a thermodestabilizing modification of the duplex in the antisense strand.

[0465] In some embodiments, antisense strand comprises at least one 2'-fluoro nucleotide adjacent to destabilizing modification.For example, 2'-fluoro nucleotide can be the nucleotide at the 5'-end or 3'-end of destabilizing modification, that is, the nucleotide at the -1 or +1 position from the position of destabilizing modification.In some embodiments, antisense strand comprises 2'-fluoro nucleotide at each of the 5'-end and 3'-end of destabilizing modification, that is, the -1 and +1 positions from the position of destabilizing modification.

[0466] In some embodiments, the antisense strand comprises at least two 2'-fluoro nucleotides at the 3' end of the destabilizing modification, ie, at positions +1 and +2 from the position of the destabilizing modification.

[0467] In some embodiments, the sense strand does not contain a 2'-fluoro nucleotide at a position opposite or complementary to a thermally destabilizing modification in the antisense strand of the duplex.

[0468] In some embodiments, all nucleotides in the sense strand and / or antisense strand can be modified.Each nucleotide can be modified with the same or different modifications, and these modifications can include one or more of the following: one or both of the non-linked phosphate oxygens and / or one or more of the linked phosphate oxygens; modification of the ribose sugar component, for example, the 2' hydroxyl on the ribose sugar; complete replacement of the phosphate moiety with a "dephospho" linker; modification or replacement of natural base; and replacement or modification of the ribose-phosphate backbone.

[0469] Because nucleic acids are polymers of monomers, many modifications occur at positions that are repeated within the nucleic acid, such as modifications of bases, phosphate moieties, or non-linked Os in phosphate moieties. In some cases, modifications will occur at all of the target positions in the nucleic acid, but in many cases, this will not be the case. For example, modifications can occur only at the 3'- or 5'-terminal positions, or only in terminal regions, such as positions on the terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of the chain. Modifications can occur in double-stranded regions, single-stranded regions, or both. Modifications can occur only in double-stranded regions of RNA or only in single-stranded regions of RNA. For example, phosphorothioate modifications of non-linked O positions can occur only at one or both ends, or only in terminal regions, such as positions on the terminal nucleotides or in the last 2, 3, 4, 5, or 10 nucleotides of the chain, or in double-stranded and single-stranded regions, especially at the ends. One or more 5'-ends can be phosphorylated.

[0470] For example, it may be possible to enhance stability, include specific bases in the overhang, or include modified nucleotides or nucleotide substitutes in the single-stranded overhang, e.g., the 5' overhang or the 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 can include, for example, the use of modifications at the 2' position of the ribose sugar, such as modifications known in the art, e.g., the use of deoxyribonucleotides, 2'-deoxy-2'-fluoro (2'-F) or 2'-O-methyl modifications in place of the ribose sugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. The overhang need not be homologous or orthologous to the target sequence.

[0471] In some embodiments, each residue of sense strand and / or antisense strand is independently modified with LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-methyl, 2'-O-allyl, 2'-C-allyl, 2'-deoxy or 2'-fluoro.Strands can contain two or more modifications.In some embodiments, each residue of sense strand and antisense strand is independently modified with 2'-O-methyl or 2'-fluoro.It should be understood that these modifications are separate from at least one thermostabilizing modification of the double strand present in antisense strand.

[0472] Typically, at least two different modifications are present in the sense strand and the antisense strand. These two modifications can be 2'-deoxy, 2'-O-methyl or 2'-fluoro modifications, acyclic nucleotides, etc. In some embodiments, the sense strand and the antisense strand each contain two different modified nucleotides selected from 2'-O-methyl or 2'-deoxy. In some embodiments, each residue in the sense strand and the antisense strand is independently modified with 2'-O-methyl nucleotides, 2'-deoxy nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-ON-methylacetamide (2'-O-NMA) nucleotides, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) nucleotides, 2'-O-aminopropyl (2'-O-AP) nucleotides, or 2'-ara-F nucleotides. Again, it should be understood that these modifications are in addition to at least one thermostabilizing modification of the duplex present in the antisense strand.

[0473] In some embodiments, the oligonucleotides or dsRNAs described herein contain alternating patterns of modifications, particularly in the B1, B2, B3, B1', B2', B3', and B4' regions. As used herein, the term "alternating motif" or "alternating pattern" refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of a single strand. Alternating nucleotides can refer to patterns such as every other nucleotide or every third nucleotide. For example, if A, B, and C each represent a type of modification to a nucleotide, the alternating motif can be "ABABABABABAB...," "AABBAABBAABB...," "AABAABAABAAB...," "AAABAAABAAAB...," "AAABBBAAABBB...," or "ABCABCABCABC...," etc.

[0474] The types of modifications within the alternating motif can be the same or different. For example, if A, B, C, and D each represent a type of modification on a nucleotide, the alternation pattern, i.e., the modifications at every other nucleotide, can be the same, but each of the sense or antisense strands can be selected from several possible modifications within the alternating motif, such as "ABABAB...", "ACACAC...", "BDBDBD..." or "CDCDCD...".

[0475] In some embodiments, the dsRNA comprises a modification pattern for the alternating motif on the sense strand that is shifted relative to the modification pattern for the alternating motif on the antisense strand. The shift can be such that the modified groups of nucleotides in the sense strand correspond to different modified groups of nucleotides in the antisense strand, or vice versa. For example, when the sense strand is paired with the antisense strand in the dsRNA, the alternating motif in the sense strand can begin with "ABABAB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BABABA" from 3' to 5' of the strand within the duplex region. As another example, the alternating motif in the sense strand can begin with "AABBAABB" from 5' to 3' of the strand, and the alternating motif in the antisense strand can begin with "BBAABBAA" from 3' to 5' of the strand within the duplex region, such that a complete or partial shift in the modification pattern occurs between the sense strand and the antisense strand.

[0476] In some embodiments, the oligonucleotide or dsRNA described herein contains one or more mismatches with the target or a combination thereof within the duplex. The mismatches can be present in the overhang region or in the duplex region. Base pairs can be ranked based on their tendency to promote dissociation or melting (e.g., in terms of the free energy of association or dissociation of a particular pairing) (the simplest approach is to examine each pair individually, but analyses such as next-neighbor or similarity analysis can also be used). In terms of promoting dissociation, A:U is preferred to G:C, G:U is preferred to G:C, and I:C is preferred to G:C (I = inosine). Mismatches, such as non-canonical pairings, i.e., pairings other than canonical pairings (as described elsewhere herein), are preferred to canonical (A:T, A:U, G:C) pairings, and pairings involving universal bases are preferred to canonical pairings.

[0477] In some embodiments, in order to promote dissociation of the antisense strand at the 5' end of the duplex, at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex region from the 5' end of the antisense strand can be independently selected from the group of A:U, G:U, I:C, and mismatched pairs, such as non-canonical pairings, i.e., pairings other than canonical pairings, or pairings containing universal bases.

[0478] In some embodiments, the first nucleotide from the 5' end of the antisense strand in the double-stranded region 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 from the 5' end of the antisense strand in the double-stranded region is an AU base pair. For example, the first base pair from the 5' end of the antisense strand in the double-stranded region is an AU base pair.

[0479] Without wishing to be bound by theory, the introduction of 4'- and / or 5'-modified nucleotides at the 3'-end of a phosphodiester (PO), phosphorothioate (PS) and / or phosphorodithioate (PS2) linkage of a dinucleotide at any position in a single-stranded or double-stranded nucleic acid can produce a steric effect at that internucleotide linkage, thereby protecting it from or stabilizing it against nucleases.

[0480] In some embodiments, 5'-modified nucleosides are introduced at the 3'-end of dinucleotides at any position in the sense strand and / or antisense strand.For example, 5'-alkylated nucleosides can be introduced at the 3'-end of dinucleotides at any position in the sense strand and / or antisense strand.The alkyl group at the 5' position of the ribose sugar can be racemic or enantiomerically pure R or S isomer.An exemplary 5'-alkylated nucleoside is 5'-methyl nucleoside.5'-methyl can be racemic or enantiomerically pure R or S isomer.

[0481] In some embodiments, a 4'-modified nucleoside is introduced at the 3'-end of a dinucleotide at any position in the sense strand and / or antisense strand. For example, a 4'-alkylated nucleoside can be introduced at the 3'-end of a dinucleotide at any position in the sense strand and / or antisense strand. The alkyl group at the 4'-position of the ribose sugar can be racemic or an enantiomerically pure R or S isomer. An exemplary 4'-alkylated nucleoside is a 4'-methyl nucleoside. The 4'-methyl can be racemic or an enantiomerically pure R or S isomer. Alternatively, a 4'-O-alkylated nucleoside can be introduced at the 3'-end of a dinucleotide at any position in the sense strand and / or antisense strand. The 4'-O-alkyl of the ribose sugar can be racemic or an enantiomerically pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is a 4'-O-methyl nucleoside, which can be racemic or an enantiomerically pure R or S isomer.

[0482] In some embodiments, 5'-alkylated nucleosides are introduced at any position on the sense strand or antisense strand of the sense strand and / or antisense strand, and such modifications maintain or improve the potency of the double-stranded nucleic acid. The 5'-alkyl can be racemic or enantiomerically pure R or S isomer. An exemplary 5'-alkylated nucleoside is a 5'-methyl nucleoside. The 5'-methyl can be racemic or enantiomerically pure R or S isomer.

[0483] In some embodiments, 4'-alkylated nucleosides are introduced at any position on the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the potency of dsRNA.4'-Alkyl can be racemic or enantiomerically pure R or S isomer.An exemplary 4'-alkylated nucleoside is 4'-methyl nucleoside.4'-Methyl can be racemic or enantiomerically pure R or S isomer.

[0484] In some embodiments, 4'-O-alkylated nucleosides are introduced at any position on the sense strand or antisense strand of dsRNA, and such modifications maintain or improve the potency of dsRNA. 5'-Alkyl can be racemic or enantiomerically pure R or S isomer. An exemplary 4'-O-alkylated nucleoside is 4'-O-methyl nucleoside. 4'-O-methyl can be racemic or enantiomerically pure R or S isomer.

[0485] In some embodiments, the oligonucleotides or dsRNAs described herein can include 2'-5' linkages (including 2'-H, 2'-OH, and 2'-OMe, as well as P=O or P=S). For example, 2'-5' linkage modifications can be used to enhance 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 sense strand activation by RISC. In some embodiments, the sense strand includes a 2'-5' linkage between the N-1st and N-2nd positions counting from the 5' end.

[0486] In some embodiments, the oligonucleotides or dsRNAs described herein can contain L-sugars (e.g., L-ribose, L-arabinose with 2'-H, 2'-OH, and 2'-OMe). For example, these L-sugar modifications can be used to enhance nuclease resistance, inhibit binding of the sense strand to the antisense strand, or can be used at the 5' end of the sense strand to prevent sense strand activation by RISC. In some embodiments, the sense strand contains an L-sugar nucleotide at the 5' end.

[0487] Ligand Embodiments of the various aspects described herein include ligands. Without wishing to be bound by theory, ligands alter one or more properties of the attached molecule (e.g., the oligonucleotides described herein), including, but not limited to, pharmacodynamics, pharmacokinetics, binding, absorption, intracellular distribution, cellular uptake, charge, and clearance. Ligands are routinely used in chemistry and are linked to the parent compound either directly or via an optional linking moiety or group. A preferred list of ligands includes, but is not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterol, thiocholesterol, cholic acid moieties, folic acid, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluorescein, rhodamine, coumarin, and dyes.

[0488] Preferred ligands applicable to the present invention include lipid moieties, such as cholesterol moieties (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3, 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533), aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75, 49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium-1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990, 18, 3777), polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969), adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or octadecylamine or carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923).

[0489] Ligands can include naturally occurring molecules or recombinant or synthetic molecules. Exemplary ligands include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG, e.g., PEG-2K, PEG-5K, PEG-10K, PEG-12K, PEG-15K, PEG-20K, PEG-40K), MPEG, [MPEG]2, polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, polyphosphazine, polyethyleneimine, cationic groups, spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, and quaternary polyamines. Salts, thyrotropin, melanotropin, lectins, glycoproteins, surfactant protein A, mucins, glycosylated polyamino acids, transferrin, bisphosphonates, polyglutamates, polyaspartates, aptamers, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralens, mitomas), isin C), porphyrins (e.g., TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., steroids, bile acids, cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., alpha helical peptides, amphipathic peptides, RGD peptides, cell penetrating peptides, endosomolytic / fusogenic peptides), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., naproxen), Sen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, polyvalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin, and pyridoxal), vitamin cofactors, lipopolysaccharide, p38 These include, but are not limited to, activators of MAP kinase, activators of NF-κB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, gamma interferon, natural or recombinant low-density lipoprotein (LDL), natural or recombinant high-density lipoprotein (HDL), and cell-permeation agents (a. helical cell-permeation agents).

[0490] Peptide and peptidomimetic ligands include natural or modified peptides, such as D- or L-peptides; α, β, or γ peptides; N-methylpeptides; azapeptides; peptides in which one or more amide linkages, i.e., peptide linkages, are replaced with one or more urea, thiourea, carbamate, or sulfonylurea linkages; or cyclic peptides. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Peptide or peptidomimetic ligands can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0491] Exemplary amphipathic peptides include, but are not limited to, cecropin, lycotoxin, paradaxin, buforin, CPF, bombinin-like peptide (BLP), cathelicidin, ceratotoxin, S. clava peptide, hagfish intestinal antimicrobial peptide (HFIAP), magainin, brevinin-2, dermaseptin, melittin, pleurocidin, H2A peptide, Xenopus peptide, esculentinis-1, and caerin.

[0492] As used herein, the term "endosomolytic ligand" refers to a molecule having endosomolytic properties. An endosomolytic ligand promotes the lysis of a cellular compartment, such as an endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubules, peroxisomes, or other vesicular body within a cell, and / or the transport of a composition of the invention or a component thereof therefrom into the cytoplasm of a cell. Exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly- or oligoimidazoles, linear or branched polyethyleneimines (PEI), linear and branched polyamines such as spermine, cationic linear and branched polyamines, polycarboxylates, polycations, shielded oligo- or polycations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with shielded or unshielded cationic or anionic charges, dendrimers with shielded or unshielded cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0493] Exemplary endosomolytic / fusogenic peptides include: Examples include, but are not limited to, TIFF2025534336000092.tif103161.

[0494] Without wishing to be bound by theory, fusogenic lipids fuse with membranes, thereby destabilizing them. Fusogenic lipids typically have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, 1,2-dileoyl-sn-3-phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)methanamine (DLin-k-DMA), and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-1,3-dioxolan-4-yl)ethanamine (also referred to herein as XTC).

[0495] Synthetic polymers with endosomolytic activity that can be used in the present invention are described in U.S. Patent Application Publication Nos. 2009 / 0048410, 2009 / 0023890, 2008 / 0287630, 2008 / 0287628, 2008 / 0281044, 2008 / 0281041, 2008 / 0269450, 2007 / 0105804, 20070036865, and 2004 / 0198687, the contents of which are incorporated herein by reference in their entireties.

[0496] Exemplary cell-penetrating peptides include: Examples include, but are not limited to, TIFF2025534336000093.tif96160.

[0497] Exemplary cationic groups include protonated amino groups, such as O-amine (amine = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino); aminoalkoxy, such as O(CH)n Amines (e.g., amine = NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino), amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid), and NH(CHCHNH) n Examples include, but are not limited to, those derived from CH2CH2-amine (amine = NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino).

[0498] As used herein, the term "targeting ligand" refers to any molecule that confers enhanced affinity to a selected target, such as a cell, cell type, tissue, organ, body region, or compartment, such as a cell, tissue, or organ compartment. Exemplary targeting ligands include, but are not limited to, antibodies, antigens, folic acid, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands.

[0499] Carbohydrate-based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, such as GalNAc2 and GalNAc3, D-mannose, multivalent mannose, multivalent lactose, N-acetyl-glucosamine, multivalent fucose, glycosylated polyamino acids, and lectins. The term multivalent indicates the presence of two or more monosaccharide units. Such monosaccharide subunits can be linked to each other or to a scaffold molecule by glycosidic bonds.

[0500] Some of the folic acid and folic acid analogs applicable to the present invention are described in U.S. Pat. Nos. 2,816,110, 5,552,545, 6,335,434, and 7,128,893, the contents of which are incorporated herein by reference in their entireties.

[0501] As used herein, the terms "PK-modulating ligand" and "PK modulating substance" refer to molecules that can modulate the pharmacokinetics of the oligonucleotides described herein. Exemplary PK modulating substances include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogs, peptides, protein-binding agents, vitamins, fatty acids, phenoxazines, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEG, biotin, and transthyretin-binding ligands (e.g., tetraiodothyroacetic acid, 2,4,6-triiodophenol, and flufenamic acid). Because oligomeric compounds containing several phosphorothioate intersugar linkages are also known to bind to serum proteins, short oligomeric compounds, e.g., oligonucleotides of about 5 to 30 nucleotides (e.g., 5 to 25 nucleotides, preferably 5 to 20 nucleotides, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides), containing multiple phosphorothioate linkages in their backbones, are also applicable as ligands (e.g., PK-modulating ligands) in the present invention. PK-modulating oligonucleotides can contain at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all internucleoside linkages in a PK-modulating oligonucleotide are phosphorothioate and / or phosphorodithioate linkages. In addition, aptamers that bind to serum components (e.g., serum proteins) can also be used in the present invention as PK-regulating ligands. Binding to serum components (e.g., serum proteins) can be predicted by albumin binding assays, such as those described in Oravcova, et al., Journal of Chromatography B (1996), 677:1-27.

[0502] When there are two or more ligands, these ligands can all have the same property, or all have different properties, or some ligands can have the same property, while other ligands have different properties.For example, ligands can have targeting property, endosomolytic activity, or PK adjusting property.In a preferred embodiment, all ligands have different properties.

[0503] In some embodiments of any one of the above aspects, the ligand has a structure shown in any of Formulas (IV)-(VII): TIFF2025534336000094.tif82157In formula, q 2A , q 2B , q 3A , q 3B , q4 A , q 4B , q 5A , q 5B and q 5C represents, independently at each occurrence, 0 to 20, and the repeating units can be the same or different; P 2A , P 2B , P 3A , P 3B , P 4A , P 4B , P 5A , P 5B , P 5C , T 2A , T 2B , T 3A , T 3B , T 4A , T 4B , T 5A , T 5B , T 5C each, independently at each occurrence, is absent, CO, NH, O, S, OC(O), NHC(O), CH, CHNH, or CHO; Q 2A , Q 2B , Q 3A , Q 3B , Q 4A , Q 4B , Q 5A , Q 5B, Q 5C is, independently at each occurrence, absent, alkylene, or substituted alkylene, wherein one or more methylenes are selected from O, S, S(O), SO, N(R N ), C(R')=C(R''), C≡C or C(O), R 2A , R 2B , R 3A , R 3B , R 4A , R 4B , R 5A , R 5B , R 5C each independently at each occurrence is absent, or is selected from the group consisting of NH, O, S, CH2, C(O)O, C(O)NH, NHCH(R a )C(O), -C(O)-CH(R a )-NH-, CO, CH=NO, TIFF2025534336000095.tif18153 or heterocyclyl, L 2A , L 2B , L 3A , L 3B , L 4A , L 4B , L 5A , L 5B and L 5C represents a ligand, i.e., each occurrence independently a monosaccharide (e.g., GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; R a is H or an amino acid side chain.

[0504] In some embodiments of any one of the above aspects, the ligand is a ligand of formula (VII): TIFF2025534336000096.tif40128, L 5A , L 5B and L 5C represents a monosaccharide, such as a GalNAc derivative.

[0505] Exemplary ligands include, but are not limited to, the following: TIFF2025534336000097.tif73128TIFF2025534336000098.tif196118TIFF2025534336000099.tif180147.

[0506] In some embodiments of any one of the aspects described herein, the ligand is a ligand described in U.S. Pat. No. 5,994,517 or U.S. Pat. No. 6,906,182, the contents of each of which are incorporated herein by reference in their entirety.

[0507] In some embodiments, the ligand can be a triantennary ligand as described in Figure 3 of U.S. Patent No. 6,906,182. For example, the ligand is selected from the following triantennary ligands: TIFF2025534336000100.tif193143

[0508] In some embodiments, the ligand can be, for example, a ligand described in Figures 4A and 4B of US2021 / 0123048, the contents of which are incorporated herein by reference in their entirety.

[0509] In some embodiments of any one of the aspects described herein, the ligand TIFF2025534336000101.tif171128TIFF2025534336000102.tif59128 (where n is 0 to 10, for example, n is 1 or 4), It could be TIFF2025534336000103.tif130144.

[0510] Note that when two or more ligands are present, they can be the same or different. Thus, in some embodiments of any one of the aspects described herein, all of the ligands are the same. In other embodiments of any one of the aspects described herein, the ligands are different.

[0511] Exemplary ligands include, but are not limited to, peptides, centilins, antibodies, antibody fragments, T cell targeting ligands, B cell targeting ligands, cancer cell targeting ligands (DUPA, folic acid, RGD), spleen targeting functionality, lung targeting functionality, bone marrow targeting functionality, anti-CD4 antibodies, anti-CD117 antibodies, phage display peptides, cell penetrating peptides (CPPs), itegrin ligands, polyanionic ligands, polycationic ligands, carbohydrates (GalNAc, mannose, mannose-6 phosphate, fucose, glucose, monovalent and multivalent), kidney targeting ligands, blood-brain barrier (BBB) ​​penetrating ligands, lipids, and amino acids (L-amino acids, D-amino acids, β-amino acids).

[0512] In some embodiments, the ligand comprises a lipophilic group. For example, the ligand is C 6~30 Aliphatic group or C 10~30 In some embodiments, the ligand is C 10~30 Alkyl, C 10~30 Alkenyl or C 10~30 It is an alkynyl group. For example, the ligand is a linear or branched hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. In some embodiments, the ligand is a linear hexyl, octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, or tetracosyl group. For example, the ligand is a linear hexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, icosyl, or docosyl group. For example, the ligand is a linear hexyl, octyl, decyl, dodecyl, hexadecyl, octadecyl, icosyl, or docosyl group. For example, the ligand is a linear hexadecyl group. In another example, the ligand is a linear docosyl group.

[0513] In some embodiments of any one of the aspects described herein, the ligand is selected from the group consisting of the ligands shown in Figures 25A-25D.

[0514] Linker Embodiments of the various aspects described herein include a linker. As used herein, the term "linker" refers to an organic moiety that connects two parts of a compound. A linker is typically a direct bond or an atom such as oxygen or sulfur, NR 1 , C(O), C(O)O, C(O)NR 1 , SO, SO2, SO2NH, or other units or chains of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, aryl alkyl, aryl alkenyl, aryl alkynyl, heteroaryl alkyl, heteroaryl alkenyl, heteroaryl alkynyl, heterocyclyl alkyl, heterocyclyl alkenyl, heterocyclyl alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylaryl alkyl, alkylaryl alkenyl, alkylaryl alkynyl, alkenylaryl alkyl, alkenylaryl alkenyl, alkenylaryl alkynyl, alkynylaryl alkyl, alkynylaryl alkenyl, alkynylaryl alkynyl, alkylheteroaryl alkyl, alkylheteroaryl alkenyl, alkylheteroaryl alkynyl, alkenyl hetero and alkylaryl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, wherein one or more methylenes are selected from O, S, S(O), SO, N(R LL)2, C(O), a cleavable linking group, a substituted or unsubstituted aryl, a substituted or unsubstituted heteroaryl, or a substituted or unsubstituted heterocyclic group; R LL is hydrogen, acyl, an aliphatic group, or a substituted aliphatic group.

[0515] In some embodiments, the linker is a cleavable linker. A cleavable linker is one that releases the two parts held together by the linker depending on a process within the target cell, such as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by a specific enzyme (e.g., a protease) present within the cell. Thus, the cleavable linker allows the two parts to be released in their original form after entry into the cell and processing within the target cell. Cleavable linkers include, but are not limited to, those with a bond that can be cleaved by an enzyme (e.g., a peptide linker), a bond that can be cleaved under reducing conditions (e.g., a disulfide linker), or a bond that can be cleaved under acidic conditions (e.g., a hydrazone and a carbonate).

[0516] Generally, a cleavable linker comprises at least one cleavable linking group. A cleavable linking group is a linking group that is sufficiently stable outside a cell but that is cleaved upon entry into a target cell to release the two moieties that the linker joins together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times faster, preferably at least 100 times faster, inside the target cell or under a first reference condition (which can be selected, for example, to mimic or represent intracellular conditions) than in the subject's blood or serum or under a second reference condition (which can be selected, for example, to mimic or represent conditions found in blood or serum).

[0517] Cleavable linkers are sensitive to cleavage agents, such as pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are found to be more prevalent or at higher levels or activity inside cells than in serum or blood. Examples of such degradative agents include redox agents that are selective for a specific substrate or that do not have substrate specificity, such as oxidases or reductases, or reducing agents such as mercaptans present in cells that can degrade redox-cleavable linkers by reduction; esterases; agents that can create endosomes or acidic environments, such as those that result in a pH of 5 or less; enzymes that can hydrolyze or degrade acid-cleavable linkers by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0518] Cleavable linkers, such as disulfide bonds, can be pH-sensitive. While the pH of human serum is 7.4, the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes are more acidic, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH, around 5.0. Some linkers will have cleavable linkers that are cleaved at a suitable pH, thereby releasing the cationic lipid from the ligand intracellularly or to a desired compartment of the cell.

[0519] The linker can include a cleavable linker that can be cleaved by a specific enzyme. The type of cleavable linker incorporated into the linker can depend on the target cell. For example, a liver-targeting ligand can be linked to a cationic lipid via a linker containing an ester group. Because liver cells are rich in esterases, the linker will be cleaved more efficiently in liver cells than in cell types that are not rich in esterases. Other cell types rich in esterases include lung, renal cortex, and testicular cells. Linkers containing peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synovial cells.

[0520] In general, the suitability of a candidate cleavable tether can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate tether. It may also be desirable to test the candidate cleavable tether for its ability to resist cleavage in blood or when contacted with other non-target tissues. Thus, the relative susceptibility to cleavage can be determined between first conditions selected to indicate cleavage in target cells and second conditions selected to indicate cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to perform initial evaluations under cell-free or cell culture conditions and confirm them with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2-fold, 4-fold, 10-fold, or 100-fold faster in cells (or under in vitro conditions selected to mimic intracellular conditions) than in blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0521] One class of cleavable linkers are redox-cleavable linkers that are cleaved upon reduction or oxidation and can be used in accordance with the present invention. One example of a reductively cleavable linker is a disulfide linker (-SS-).

[0522] Phosphate-based cleavable linkers that may be used in the linkers of the present invention are cleaved by an agent that degrades or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group within a cell is an enzyme, such as a phosphatase, within the cell. An example of a phosphate-based linker is -OP(O)(OR k )-O-, -OP(S)(OR k )-O-, -OP(S)(SR k )-O-, -SP(O)(OR k )-O-, -OP(O)(OR k )-S-, -SP(O)(OR k )-S-, -OP(S)(OR k)-S-, -SP(S)(OR k )-O-, -OP(O)(R k )-O-, -OP(S)(R k )-O-, -SP(O)(R k )-O-, -SP(S)(R k )-O-, -SP(O)(R k )-S-, -OP(S)(R k )-S-, wherein R k independently for each occurrence: hydrogen, C 1~20 Alkyl, C 1~20 Haloalkyl, C 6~10 Aryl, C 7~12 It may be aralkyl. Preferred embodiments are -OP(O)(OH)-O-, -OP(S)(OH)-O-, -OP(S)(SH)-O-, -SP(O)(OH)-O-, -OP(O)(OH)-S-, -SP(O)(OH)-S-, -OP(S)(OH)-S-, -SP(S)(OH)-O-, -OP(O)(H)-O-, -OP(S)(H)-O-, -SP(O)(H)-O-, -SP(S)(H)-O-, -SP(O)(H)-S-, and -OP(S)(H)-S-. One preferred embodiment is -OP(O)(OH)-O-. These candidates can be evaluated using methods similar to those described above.

[0523] Acid-cleavable linkers that can be used in the linkers of the present invention are linkers that are cleaved under acidic conditions. In preferred embodiments, acid-cleavable linkers are cleaved in an acidic environment with a pH of about 6.5 or less (e.g., about 6.0, 5.5, 5.0, or less) or by an agent, such as an enzyme, that can act as a general acid. In cells, certain low-pH organelles, such as endosomes and lysosomes, can provide a cleavage environment for acid-cleavable linkers. Examples of acid-cleavable linkers include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid-cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). In a preferred embodiment, the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group, such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0524] Ester-based cleavable linkers that can be used in the linkers of the present invention are cleaved intracellularly by enzymes such as esterases and amidases. Examples of ester-based cleavable linkers include, but are not limited to, esters of alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0525] Peptide-based cleavable linkers that can be used in accordance with the present invention are cleaved intracellularly by enzymes such as peptidases and proteases. Peptide-based cleavable linkers are peptide bonds formed between amino acids to give oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to give peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable linkers have the general formula -NHCHR A C(O)NHCHR B C(O)—, where R A and R B are the R groups of two adjacent amino acids.

[0526] In some embodiments of any one of the aspects described herein, the linker is a hydrophobic linker. For example, the linker includes an aliphatic, cycloaliphatic, and / or aromatic moiety. In some embodiments, the linker is a hydrophilic linker. For example, the linker includes polyethylene glycol. For example, the linker is -(CH2CH2O) w -, where w is an integer. In some embodiments, w is an integer between 1 and 1000. For example, w is an integer between 2 and 500, e.g., w is 5, 10, 15, 20, 25, 30, 35, 40, 50, 100, 150, 200, 250, 300, 350, 400, or 500.

[0527] Oligonucleotide Modification In some embodiments of any one of the above aspects, the oligonucleotide can include one or more, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more, modified internucleoside linkages. For example, the oligonucleotide can include 1, 2, 3, 4, 5, or 6 modified internucleoside linkages. For example, the oligonucleotide includes 1, 2, 3, or 4 modified internucleoside linkages. In some embodiments, the oligonucleotide includes at least two modified internucleoside linkages within the first five nucleotides counting from the 5' end of the oligonucleotide and further includes at least two modified internucleoside linkages within the first five nucleotides counting from the 3' end of the oligonucleotide. For example, the oligonucleotide includes modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the oligonucleotide.

[0528] In some embodiments of any one of the above aspects, the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 or more, phosphorothioate internucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, 4, 5, or 6 phosphorothioate internucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, or 4 phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 5' end of the oligonucleotide and further comprises at least two phosphorothioate internucleoside linkages within the first five nucleotides counting from the 3' end of the oligonucleotide. For example, the oligonucleotide comprises modified internucleoside linkages between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 5' end of the oligonucleotide, and between nucleotides 1 and 2 and between nucleotides 2 and 3 counting from the 3' end of the oligonucleotide.

[0529] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (II), a nucleoside having a modified sugar. By "modified sugar" is meant a sugar or moiety other than a 2'-deoxy (i.e., 2'-H) ribose sugar or a 2'-OH ribose sugar. Exemplary nucleotides containing modified sugars include 2'-F ribose, 2'-OMe ribose, 2'-O,4'-C-methylene ribose (locked nucleic acid, LNA), anhydrohexitol (1,5-anhydrohexitol nucleic acid, HNA), cyclohexene (cyclohexene nucleic acid, CeNA), 2'-methoxyethyl ribose, 2'-O-allyl ribose, 2'-C-allyl ribose, 2'-ON-methylacetamido (2'-O-NMA) ribose, 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) ribose, 2'-O-aminopropyl (2'-O-AP) ribose, 2'-F arabinose (2'-ara-F), threose (threose nucleic acid, TNA), and 2,3-dihydroxylpropyl (glycol nucleic acid, GNA). It should be noted that the nucleoside having the modified sugar can be present at any position in the oligonucleotide.

[0530] In some embodiments, the oligonucleotide further comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoro (2'-F) nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-F nucleotides. Note that the 2'-F nucleotides can be present at any position in the oligonucleotide.

[0531] In some embodiments, the oligonucleotide comprises, for example, exclusively, nucleosides of Formula (II) and 2'-F nucleosides.

[0532] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-OMe nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-OMe nucleotides. Note that the 2'-OMe nucleotides can be present at any position in the oligonucleotide.

[0533] In some embodiments, the oligonucleotide comprises, for example, exclusively, nucleosides of formula (II) and 2'-OMe nucleosides. In other embodiments, the oligonucleotide comprises, for example, exclusively, nucleosides of formula (II), 2'-OMe nucleosides, and 2'-F nucleosides.

[0534] In some embodiments, the oligonucleotide further comprises at least one, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-deoxy, e.g., 2'-H nucleotides. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-deoxy, e.g., 2'-H nucleotides....

Claims

1. A double-stranded RNA (dsRNA) comprising an antisense strand and a sense strand complementary to the antisense strand, wherein the antisense strand comprises a first ligand at its 3' end, the antisense strand comprises at least one nuclease-resistant modification at its 3' end and at least one nuclease-resistant modification at its 5' end, and the dsRNA has a double-stranded region of at least about 15 base pairs.

2. 2. The dsRNA of claim 1, wherein the sense strand comprises at least one nuclease-resistant modification at its 5'-end, and optionally, the sense strand comprises at least one nuclease-resistant modification at its 3'-end and at least one nuclease-resistant modification at its 5'-end.

3. 2. The dsRNA of claim 1, wherein the at least one nuclease-resistant modification is a modified internucleoside linkage, a modified sugar moiety, or a modified nucleobase, and optionally, the at least one nuclease-resistant modification is a phosphorothioate internucleoside linkage, a phosphorodithioate internucleoside linkage, a 2'-5' linked nucleotide, or an L-nucleotide.

4. the antisense strand comprises at least two, e.g., three, four, five, or six or more, phosphorothioate internucleoside linkages, and optionally a. the antisense strand comprises a phosphorothioate internucleoside linkage between the first and second nucleotides from the 3' end of the antisense strand and a phosphorothioate internucleoside linkage between the first and second nucleotides from the 5' end of the antisense strand; or b. the antisense strand contains phosphorothioate internucleoside linkages between the first and second and between the second and third nucleotides, counting from the 3' end of the strand, and phosphorothioate internucleoside linkages between the first and second and between the second and third nucleotides, counting from the 5' end of the strand; or c. the antisense strand contains phosphorothioate internucleoside linkages between the first and second, second and third, and third and fourth nucleotides, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second nucleotides, counting from the 5' end of the strand; or d. the antisense strand contains a phosphorothioate internucleoside linkage between the first and second nucleotides, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second nucleotides and between the second and third nucleotides, counting from the 5' end of the strand; or e. the antisense strand contains a phosphorothioate internucleoside linkage between the first and second nucleoside residues, counting from the 3' end of the strand, and a phosphorothioate internucleoside linkage between the first and second nucleoside residues, between the second and third nucleoside residues, and between the third and fourth nucleoside residues, counting from the 5' end of the strand; The dsRNA of claim 1.

5. the sense strand comprises at least one, e.g., two, three, or four or more, phosphorothioate internucleoside linkages, and optionally a. the sense strand contains a phosphorothioate internucleoside linkage between the first and second nucleotides from the 5' end of the strand; b. the sense strand contains phosphorothioate internucleoside linkages between the first and second positions counting from the 5' end of the strand and between the first and second positions counting from the 3' end of the strand; c. the sense strand contains phosphorothioate internucleoside linkages between the first and second and between the second and third nucleotides, counting from the 5' end of the strand; or d. the sense strand contains phosphorothioate internucleoside linkages between the first and second and between the second and third positions, counting from the 5' end of the strand, and between the first and second and between the second and third positions, counting from the 3' end of the strand; The dsRNA of claim 1.

6. 2. The dsRNA of claim 1, wherein the first ligand is linked to the 3'-hydroxyl of the first nucleotide counting from the 3' end of the antisense strand.

7. The first ligand is linked to the 3' end of the antisense strand via a linker, optionally the linker being a hydrophobic linker or a hydrophilic linker (e.g., polyethylene glycol, e.g., -(CH 2 CH 2 O) w -, where w is an integer, optionally w is an integer from 1 to 1000, for example, an integer from 2 to 500.

8. 7. The dsRNA of claim 6, wherein the linker is linked to the 3'-end of the antisense strand via a phosphodiester internucleoside linkage or a phosphorothioate internucleoside linkage.

9. 9. The dsRNA of claim 7 or 8, wherein the linker is about 5 angstroms to about 250 angstroms in length and / or the linker has a chain length of at least 6 atoms.

10. 10. The dsRNA of any one of claims 7 to 9, wherein the linker comprises a hydrophobic carrier tethered to a carrier, optionally wherein the carrier comprises a hydrogen bond acceptor, and optionally wherein the carrier comprises a pyrrolidine ring.

11. 2. The dsRNA of claim 1, wherein the antisense strand is at least about 17 nucleotides in length, and optionally the antisense strand is about 19, about 20, about 21, about 22, about 23, about 24, about 25, or about 26 nucleotides in length, and preferably the antisense strand is about 22, about 23, or about 25 nucleotides in length.

12. 2. The dsRNA of claim 1, wherein the sense strand is at least about 15 nucleotides in length, and optionally the sense strand is about 19, about 20, about 21, about 22, about 23, about 24, or about 25 nucleotides in length, and preferably the sense strand is about 21 nucleotides in length.

13. (a) the sense strand is 15 nucleotides in length and the antisense strand is 18, 19, 20, 21, or 22 (e.g., 20) nucleotides in length; (b) the sense strand is 19 nucleotides in length and the antisense strand is 19, 20, or 21 nucleotides in length; (c) the sense strand is 20 nucleotides in length and the antisense strand is 20, 21, or 22 nucleotides in length; (d) the sense strand is 21 nucleotides in length and the antisense strand is 21, 22, or 23 nucleotides in length; or (e) the sense strand is 20-24 (e.g., 22) nucleotides in length and the antisense strand is 34-38 (e.g., 36) nucleotides in length; The dsRNA of claim 1.

14. 2. The dsRNA of claim 1, having a double-stranded region of at least about 15 base pairs, optionally about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 base pairs or more, and preferably about 21 base pairs.

15. 2. The dsRNA of claim 1, wherein the sense strand is about 21 nucleotides in length, the antisense strand is about 21, about 22, about 23, about 24 or about 25 nucleotides in length, and the dsRNA comprises a double-stranded region of at least 18, for example, 19, 20 or 21 base pairs.

16. dsRNA according to any one of the preceding claims, wherein the dsRNA comprises at least one single-stranded overhang comprising 1 to 5 nucleotides (e.g., 1 or 2 nucleotides), and optionally the antisense strand comprises a single-stranded overhang at its 3' end.

17. 2. The dsRNA of claim 1, wherein the dsRNA comprises at least one blunt end, and optionally the antisense strand comprises a blunt end at its 5' end.

18. 18. The dsRNA of claim 16 or 17, wherein the antisense strand comprises at least one nuclease-resistant modification in the single-stranded overhang, and / or the antisense strand comprises at least one phosphorothioate internucleoside linkage in the single-stranded overhang.

19. 2. The dsRNA of claim 1, wherein the ligand comprises GalNAc.

20. The ligand is (wherein n is 0 to 10, for example, n is 1 or 4), and optionally, the first ligand is The dsRNA according to any one of the preceding claims,

21. dsRNA according to any one of the preceding claims, wherein the dsRNA comprises a second ligand, and optionally, the second ligand is linked to the sense strand.

22. 22. The dsRNA of claim 21, wherein the second ligand is a PK modulator, a targeting ligand or an endosomolytic ligand, and optionally, the second ligand is a PK modulator.

23. 23. The dsRNA of any one of claims 21 to 22, wherein the second ligand binds to a serum protein, such as serum albumin.

24. 24. The dsRNA of any one of claims 21-23, wherein the second ligand comprises iodipamide, azapropazone, indomethacin, tiburon (TIB), 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), DIS, oxyphenbutazone, phenylbutazone, warfarin, indoxyl sulfate, diflunisal, halothane, ibuprofen, diazepam, propofol, or any combination thereof, optionally wherein the second ligand comprises ibuprofen.

25. 25. The dsRNA of any one of claims 21 to 24, wherein the first ligand comprises GalNAc and the second ligand comprises ibuprofen, or the first ligand is GalNAc and the second ligand is a mannose receptor targeting ligand (e.g., multivalent mannose), or the first ligand is GalNAc and the second ligand is a folate ligand.

26. 26. The dsRNA of any one of claims 21 to 25, comprising at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more 2'-fluoronucleotides.

27. the antisense strand comprises at least one, e.g., 2, 3, 4, or 5 or more 2'-fluoro nucleotides, and optionally a. the antisense strand contains 2'-fluoro nucleotides at positions 2, 14, and 16, counting from the 5' end of the antisense strand; b. the antisense strand contains 2'-fluoro nucleotides at positions 2, 6, 14, and 16, counting from the 5' end of the antisense strand; c. the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 9, 14, and 16, counting from the 5' end of the antisense strand; or d. the antisense strand contains 2'-fluoronucleotides at positions 2, 6, 8, 9, 14, and 16, counting from the 5' end of the antisense strand; 27. The dsRNA of any one of claims 21 to 26.

28. the antisense strand comprises at least one, e.g., 2, 3, 4, or 5 or more 2'-fluoro nucleotides, and optionally a. the sense strand contains 2'-fluoronucleotides at positions 7, 9, and 11 counting from the 5' end of the sense strand or at positions 11, 13, and 15 counting from the 3' end of the sense strand; b. the sense strand contains 2'-fluoronucleotides at positions 7, 9, 10, and 11 counting from the 5' end of the sense strand or positions 11, 12, 13, and 15 counting from the 3' end of the sense strand; or c. the sense strand contains 2'-fluoronucleotides at positions 9, 10, and 11 counting from the 5' end of the sense strand or positions 11, 12, and 13 counting from the 3' end of the sense strand; 28. The dsRNA of any one of claims 21 to 27.

29. the antisense strand comprises at least one, e.g., 2, 3, 4, 5, 6, or 7 or more DNA nucleotides, and optionally a. the antisense strand comprises DNA nucleotides at positions 2, 5, 7, and 12, counting from the 5' end of the antisense strand, and optionally, the antisense strand further comprises a 2'-fluoro nucleotide at position 14, counting from the 5' end of the antisense strand; or b. the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, and 14, counting from the 5' end of the antisense strand; or c. the antisense strand comprises DNA nucleotides at positions 2, 5, 7, 12, 14, and 16, counting from the 5' end of the antisense strand; The dsRNA of any one of claims 21 to 28.

30. 30. The dsRNA of any one of claims 21 to 29, wherein the antisense strand comprises at least one 2'-OMe nucleotide, and optionally, all remaining nucleotides in the antisense strand are 2'-OMe nucleotides.

31. 31. The dsRNA of any one of claims 21 to 30, wherein the sense strand comprises at least one 2'-OMe nucleotide, and optionally, all remaining nucleotides in the sense strand are 2'-OMe nucleotides.

32. 32. The dsRNA of any one of claims 21 to 31, wherein the antisense strand comprises a phosphate group or a phosphate analog or a derivative thereof at its 5' end, and optionally, the antisense strand comprises a vinyl phosphonate (e.g., E-vinyl phosphonate) group at its 5' end.

33. a. the antisense strand comprises at least one, e.g., 2, 3, 4, or 5 or more LNA or BNA nucleotides; b. the sense strand comprises at least one, e.g., 2, 3, 4, or 5 or more LNA or BNA nucleotides; c. the antisense strand comprises at least one, e.g., 2, 3, 4, or 5 or more CeNA nucleotides; d. the antisense strand contains at least one, e.g., two, three, four, or five or more, thermostabilizing modifications; e. the sense strand comprises at least one, e.g., two, three, four, or five or more, thermostabilizing modifications; f. the antisense strand contains at least one, e.g., 2, 3, 4, or 5 or more abasic nucleotides; g. the sense strand contains at least one, e.g., 2, 3, 4, or 5 or more abasic nucleotides; h. the antisense strand comprises at least one, e.g., 2, 3, 4, or 5 or more 2'-deoxynucleotides; i. the sense strand comprises at least one, e.g., 2, 3, 4, or 5 or more 2'-deoxynucleotides; j. the antisense strand comprises at least one acyclic (e.g., 2, 3, 4, or 5 or more) nucleotides, such as unlocked nucleic acid (UNA) or glycol nucleic acid (GNA)); k. the sense strand contains at least one, e.g., 2, 3, 4, or 5 or more acyclic (e.g., unlocked nucleic acid (UNA) or glycol nucleic acid (GNA)) nucleotide; and / or l. the antisense strand comprises at least one thermodestabilizing modification, and optionally the antisense strand comprises at least one thermodestabilizing modification in the seed region of the antisense strand (i.e., positions 2 to 9, e.g., 6, 7, or 8, counting from the 5' end), and optionally the thermodestabilizing modification is an abasic nucleotide, a 2'-deoxynucleotide, an acyclic nucleotide (e.g., unlocked nucleic acid (UNA), glycol nucleic acid (GNA), or (S)-glycol nucleic acid (S-GNA)), a 2'-5' linked nucleotide (3'-RNA), a threose nucleotide (TNA), a 2' gem Me / F nucleotide, or a mismatch with the opposite nucleotide in the other strand; The dsRNA of any one of claims 21 to 32.

34. Compounds of formula (I): During the ceremony, B is an optionally modified nucleobase; X S O, CH 2 , S, or NH; R 2 is a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), a phosphate group, a reactive phosphorus(III) group, a ligand, or a linker covalently bonded to one or more ligands; R 3 is a reactive phosphorus(III) group, hydroxyl, protected hydroxyl, halogen, optionally substituted C 2～30 Alkynyl, optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), a phosphate group, a ligand, or a linker covalently bonded to one or more ligands; R 4 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 Is it an alkoxy? or R 4 and R 2 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N (R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 ) - and R 10 and R 11 are independently H, optionally substituted C 1 ~C 6 Alkyl, optionally substituted C 2 ~C 6 Alkenyl or optionally substituted C 2 ~C 6 is alkynyl, R 12 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, v is 1, 2, or 3; R 5 -L 1 -R H , -ON(R 13 ) R 14 and where X P is a phosphate group, L 1 is a bond, -L 3 -, C 1～30 Alkylene, C 2～30 Alkenylene, C 2～30 Alkynylene, *-L 3 -C 1～30 Alkylene *-L 3 -C 2～30 Alkenylene, or *-L 3 -C 2～30 is alkynylene, L 3 は、-O-、-N(R L3 )-、-S-、-C(O)-、-S(O)-、-S(O) 2 -、-P(X L3 )(Y L3 R L3B )- where R L3 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, X L2 is O or S, Y L3 is O, S, NH, or a bond, R L3B is H or optionally substituted alkyl, * indicates R H is a bond to and R H is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, said heterocyclyl being optionally substituted with 1, 2, 3 or 4 independently selected substituents, provided that said heterocyclyl contains at least one nitrogen atom; or R H teeth where X is O, NR L , S, or CH 2 and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar; R 13 and R 14 independently, -L 2 -R H2 or C 1 ~C 6 alkyl, where L 2 is the linker, R H2 is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, said heterocyclyl being optionally substituted with 1, 2, 3 or 4 independently selected substituents; However, R 13 and R 14 At least one of them is -L 2 -R H2 and R 2 and R 3 and only one of the groups is a reactive phosphorus(III) group; R 5 is R 4 and R 2 together form 4'-C (R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', and is not morpholin-4-yl.

35. R 5 Ga-L 1 -R H and optionally, L 1 L 3 or C 1～30 alkylene, and optionally, L 3 35. The compound of claim 34, wherein is O.

36. R H is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1 or 2 additional heteroatoms independently selected from N, O and S, and optionally a.R H but where X is O, NR L , S, or CH 2 and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylamino alkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars; and optionally, X is O or NR L and optionally, R L is hydrogen, a ligand, or a linker covalently bonded to one or more independently selected ligands; b. R H but and optionally X is O or the compound of claim 123, wherein X is NR L and optionally, R L is hydrogen, a ligand, or a linker covalently bonded to one or more independently selected ligands; 36. The compound of claim 35.

37. R 5 -ON (R 13 ) R 14 and optionally, R 13 and R 14 One of them is -L 2 -R H2 and optionally, L 2 35. The compound of claim 34, wherein is a bond or an optionally substituted alkylene.

38. R 13 and R 14 One of the groups is -(CH 2 ) m -R H2 or and optionally, R H2 is an optionally substituted 6-membered heterocyclyl containing a nitrogen atom and 0, 1 or 2 additional heteroatoms independently selected from N, O and S, and optionally a.R H2 but where X is O, NR L , S, or CH 2 and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with aliphatic and aromatic alkyls, alkyl esters, alkylamines, dimethylamino alkyls, alkyl ethers, alkyl thioethers, heteroaromatic alkyls, allyls, vinyls, disulfides, oximes, ketones, acetals, hemiacetals, cleavable peptides, or cleavable sugars; and optionally, X is O or NR L and optionally, R L is hydrogen, a ligand, or a linker covalently bonded to one or more independently selected ligands; or b. R H2 but where X is O, NR L , S, or CH 2 and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylamino alkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar; 38. The compound of claim 34 or 37.

39. R 13 and R 14 one of which is optionally substituted C 1 ~C 6 alkyl, and optionally R 13 and R 14 39. The compound of any one of claims 34-38, wherein one of: is methyl.

40. X S is O or CH 2 and optionally, X S The compound of any one of claims 34 to 39, wherein is O.

41. R 3 is a reactive phosphorus(III) group, hydroxyl, or protected hydroxyl, and optionally, R 3 is a reactive phosphorus(III) group.

42. R 2 is hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), or -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), and optionally, R 2 is hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino, and optionally R 2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy, and optionally, R 2 42. The compound of any one of claims 34-41, wherein is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

43. R 2 and R 4 together, 4'-C (R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', and optionally, R 2 and R 4 together, 4'-C (R 10 R 11 ) v -Y-2', where v is 1 or 2, and optionally R 10 and R 11 one of which is H and the other is independently H or optionally substituted C 1 ~C 6 alkyl, and optionally R 2 and R 4 But together, 4'-CH 2 43. The compound of any one of claims 34 to 42, wherein:

44. R 2 is a reactive phosphorus(III) group, hydroxyl, or protected hydroxyl, and optionally, R 2 is a reactive phosphorus(III) group.

45. R 3 is hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), or -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), and optionally, R 3 is hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino, and optionally R 3 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy, and optionally, R 3 45. The compound of claim 44, wherein is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

46. R 4 46. ​​The compound of any one of claims 34-42, 44 or 45, wherein is H.

47. Formulas (IA) to (ID): (In the formula, n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, or dialkylamino; R 3 is a reactive phosphorus(III) group, a hydroxyl, a protected hydroxyl or a reactive phosphorus(III) group, R 4 is hydrogen or R 2 and R 4 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2') 35. The compound of claim 34, selected from:

48. X S 48. The compound of claim 47, wherein is O.

49. R 3 is a reactive phosphorus(III) group, hydroxyl or protected hydroxyl, and optionally, R 3 is a reactive phosphorus(III) group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

50. R 2 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy (e.g., methoxy, 2-methoxyethoxy), or alkoxyalkyl (e.g., 2-methoxyethyl), and optionally R 2 50. The compound of any one of claims 47-49, wherein is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

51. R 4 51. The compound of any one of claims 47-50, wherein

52. R 2 and R 4 together, 4'-C (R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', and optionally, R 10 and R 11 is H, and the other is independently H or optionally substituted C 1 ~C 6 alkyl, and optionally R 2 and R 4 But together, 4'-CH 2 51. The compound of any one of claims 47 to 50, wherein:

53. R 13 and R 14 One of them is and R 13 and R 14 The other of these is C 1 ~C 6 Alkyl, 53. The compound of any one of claims 47 to 52, wherein

54. R 13 and R 14 One of them is C 1 ~C 6 54. The compound of any one of claims 34 to 53, which is alkyl (e.g., methyl).

55. X is O, S, or CH 2 , NH or NR L and optionally, R L is a ligand or a linker covalently attached to one or more independently selected ligands.

56. Compounds of formula (IE): (In the formula, R 3 is a reactive phosphorus(III) group, a hydroxyl, or a protected hydroxyl; R 5 -L 1 -R H and X S , B, Y, R 10 and R 11 is as defined in claim 111) 35. The compound of claim 34, wherein:

57. Formula (I-Ea), (IE 1 ) or (IE 2 ) compounds: (In the formula, n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); X is O, NR L , S, or CH 2 and R L is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar) and Optionally, a compound of formula (I-Eb) or (I-Ec):

57. The compound of claim 56, wherein:

58. X is O or CH 2 and optionally, X is O.

59. 59. The compound of any one of claims 56-58, wherein Y is O.

60. R 10 One of them is H and the other is H or C 1～6 60. The compound of any one of claims 56 to 59, which is alkyl (e.g., methyl).

61. X S The compound of any one of claims 56-60, wherein

62. R 3 is a reactive phosphorus(III) group, hydroxyl or protected hydroxyl, and optionally, R 3 is a reactive phosphorus(III) group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(β-thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

63. Formulas (I-Ed), (I-Ee), (IE 3 ) or (IE 4 ) compounds: wherein n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R L is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar) and Optionally, a compound of formula (I-Ef) or (I-Eg):

57. The compound of claim 56, wherein:

64. An oligonucleotide prepared using a compound according to any one of claims 34 to 63.

65. An oligonucleotide comprising at least one nucleoside of formula (II): During the ceremony, B is an optionally modified nucleobase; X S O, CH 2 , S, or NH; R 22 is a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, optionally substituted C 2～30 Alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), a ligand, a linker covalently attached to one or more ligands, or a bond to an internucleotide linkage to a subsequent nucleoside; R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, a hydroxyl, a protected hydroxyl, a halogen, an optionally substituted C 2～30 Alkynyl, optionally substituted C 1～30 Alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 2～30 Alkenyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5- to 8-membered heterocyclyl, -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), -OC 4～30 Alkyl-ON (CH 2 R 8 ) (CH 2 R 9 ), a phosphate group, a ligand, or a linker covalently bonded to one or more ligands; R 24 is hydrogen, optionally substituted C 1～6 Alkyl, optionally substituted C 2～6 Alkenyl, optionally substituted C 2～6 Alkynyl, or optionally substituted C 1～6 Is it an alkoxy? or R 22 and R 24 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', Y is -O-, -CH 2 -, -CH(Me)-, -C(CH 3 ) 2 -, -S-, -N (R 12 )-, -C(O)-, -C(S)-, -S(O)-, -S(O) 2 -, -OC(O)-, -C(O)O-, -N(R 12 )C(O)-, or -C(O)N(R 12 ) - and R 10 and R 11 are independently H, optionally substituted C 1 ~C 6 Alkyl, optionally substituted C 2 ~C 6 Alkenyl or optionally substituted C 2 ~C 6 is alkynyl, R 12 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, v is 1, 2, or 3; R 5 -L 1 -R H , -ON(R 13 ) R 14 and where X P is a phosphate group, L 1 is a bond, -L 3 -, C 1～30 Alkylene, C 2～30 Alkenylene, C 2～30 Alkynylene, *-L 3 -C 1～30 Alkylene *-L 3 -C 2～30 Alkenylene, or *-L 3 -C 2～30 is alkynylene, L 3 は、-O-、-N(R L3 )-、-S-、-C(O)-、-S(O)-、-S(O) 2 -、-P(X L3 )(Y L3 R L3B )- where R L3 is hydrogen, optionally substituted C 1～30 Alkyl, optionally substituted C 1 ~C 30 Alkoxy, C 1～4 Haloalkyl, optionally substituted C 2～4 Alkenyl, optionally substituted C 2～4 Alkynyl, optionally substituted C 1～30 Alkyl-CO 2 H, or a nitrogen protecting group, X L2 is O or S, Y L3 is O, S, NH, or a bond, R L3B is H or optionally substituted alkyl, * indicates R H is a bond to R H is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, said heterocyclyl being optionally substituted with 1, 2, 3 or 4 independently selected substituents, provided that said heterocyclyl contains at least one nitrogen atom, or R H teeth where X is O, NR L , S, or CH 2 and R L is hydrogen, a ligand, a linker covalently bonded to one or more ligands, an alkyl group functionalized with an aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar; R 13 and R 14 independently, -L 2 -R H2 where: L 2 is the linker, R H2 is a 4-8 membered heterocyclyl containing 1, 2 or 3 heteroatoms independently selected from N, O and S, said heterocyclyl being optionally substituted with 1, 2, 3 or 4 independently selected substituents; However, R 13 and R 14 At least one of them is -L 2 -R H2 and However, R 22 and R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, and R 22 and R 23 Only one of the bonds is to the internucleotide linkage to the subsequent nucleoside.

66. The nucleoside of formula (II) is represented by formulas (II-A) to (II-D): (In the formula, R 5 -L 1 -R H and R 22 is hydrogen, hydroxyl, protected hydroxyl, halogen, optionally substituted C 1～30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), amino, alkylamino, or dialkylamino; R 23 is the bond to the internucleotide linkage to the subsequent nucleoside, R 24 is hydrogen or R 22 and R 24 together form 4'-C(R 10 R 11 ) v -Y-2' or 4'-YC (R 10 R 11 ) v -2', X S , B, Y, R 10 and R 11 is as defined in claim 34) 66. The oligonucleotide of claim 65, selected from:

67. The nucleoside is a nucleoside of formula (II-Ea), (II-E') or (II-E''): (In the formula, n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); X is O, NR L , S, or CH 2 and R L is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar) and Optionally, the compound is a compound of formula (II-Eb) or (II-Ec):

67. The oligonucleotide of claim 66, wherein:

68. The nucleoside is represented by the formula (II-Ed), (II-Ee), (II-E 3 ) or (II-E 4 ) nucleosides: wherein n is 0 or an integer selected from 1 to 30 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, e.g., n is 1, 2, 3, 4, 5 or 6, preferably n is 0 or 1); R L is an alkyl group functionalized with H, a ligand, a linker covalently bonded to one or more ligands, aliphatic and aromatic alkyl, alkyl ester, alkylamine, dimethylaminoalkyl, alkyl ether, alkyl thioether, heteroaromatic alkyl, allyl, vinyl, disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptide, or cleavable sugar) and Optionally, the compound is a compound of formula (II-Ef) or (II-Eg):

68. The oligonucleotide of claim 67, wherein:

69. 69. The oligonucleotide of any one of claims 65 to 68, comprising 3 to 50 nucleotides.

70. a. contains at least one ribonucleotide; b. contains at least one 2'-deoxyribonucleotide; c. In addition to the nucleosides of formula (II), at least one nucleoside has a modified or non-natural nucleobase; d. In addition to the nucleosides of formula (II), at least one nucleoside has a modified ribose sugar; e. In addition to the nucleosides of formula (II), at least one nucleoside contains a group other than H or OH at the 2' position of the ribose sugar; f. In addition to the nucleoside of formula (II), it contains at least one nucleoside having 2'-F ribose; g. In addition to the nucleoside of formula (II), it contains at least one nucleoside having 2'-OMe ribose, h. In addition to the nucleosides of formula (II), at least one nucleoside contains a moiety other than a ribose sugar; i. comprises at least one modified internucleotide linkage; j. the internucleotide linkage to the subsequent nucleoside of Formula (II) is a modified internucleotide linkage, optionally wherein the modified internucleotide linkage is a phosphorothioate linkage; k. attached to a solid support; l. comprises at least one ligand, and / or m. containing at least one hydroxyl, phosphate, or amino protecting group; 70. The oligonucleotide of any one of claims 65 to 69.

71. 71. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein the first or second strand is the oligonucleotide of any one of claims 64-70, and optionally, the first and second strands are independently 15-25 nucleotides in length.

72. 72. The double-stranded nucleic acid of claim 71, capable of inducing RNA interference.

73. One of the first strand and the second strand is the oligonucleotide according to any one of claims 64 to 70, and the other strand has a vinylphosphonate (VP) group (e.g., *=CH-X P , X P is a phosphate group, and * is C5'), C 3～6 Cycloalkylphosphonates (e.g., cyclopropylphosphonate), monophosphates ((HO) 2 (O)PO-5'), diphosphate ((HO) 2 (O)POP(HO)(O)-O-5'), triphosphate ((HO) 2 (O)PO-(HO)(O)POP(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)PO-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)PO-5'), phosphorothiolate ((HO)2(O)PS-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidate ((HO) 2 (O)P-NH-5', (HO)(NH 2 ) (O)PO-5'), alkylphosphonate [(R P )(OH)(O)PO-5', R P is optionally substituted C 1～30 alkyl, for example, methyl, ethyl, isopropyl, or propyl)], alkyl ether phosphonates [(R P1 )(OH)(O)PO-5', R P1 is an alkoxyalkyl, e.g., methoxymethyl (CH 2 ethoxymethyl (OMe) or ethoxymethyl (HO) 2 (X)PO[-(CH 2 ) a -OP(X)(OH)-O] b -5' or (HO) 2 (X)PO[-(CH 2 ) a -P(X)(OH)-O] b -5' or (HO) 2 (X)P-[-(CH 2 ) a -OP(X)(OH)-O] b -5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH 2 ) a -OP(X)(OH)-O] b -5', H 2 N [-(CH 2 ) a -OP(X)(OH)-O] b -5', H [-(CH 2 ) a -OP(X)(OH)-O] b -5', Me 2 N [-(CH 2 ) a -OP(X)(OH)-O] b -5', HO [-(CH 2 ) a -P(X)(OH)-O] b -5', H 2 N [-(CH 2 ) a -P(X)(OH)-O] b -5', H [-(CH 2 ) a -P(X)(OH)-O] b -5', Me 2 N [-(CH 2 ) a -P(X)(OH)-O] b 73. The double-stranded nucleic acid of claim 71 or 72, comprising a vinyl phosphonate group, such as an E-vinyl phosphonate group, and a and b each independently represent 1 to 10, wherein X is O or S, and a and b each independently represent 1 to 10, and optionally the strand comprises a vinyl phosphonate group, such as an E-vinyl phosphonate group.

74. 74. The double-stranded nucleic acid of any one of claims 71 to 73, comprising a sense strand and an antisense strand, wherein the sense strand is the oligonucleotide of any one of claims 64 to 70.

75. 75. The double-stranded nucleic acid of any one of claims 71-74, wherein one or both strands have a 1-5 nucleotide overhang at their respective 5' or 3' ends, and optionally, only one strand has a 2 nucleotide overhang at its 5' or 3' end, and optionally, only one strand has a 2 nucleotide overhang at its 3' end.

76. 10. A pharmaceutical composition comprising the oligonucleotide of any one of claims 64 to 70 or the dsRNA molecule of any one of claims 1 to 33 or claims 71 to 75, alone or in combination with a pharmaceutically acceptable carrier or excipient.

77. 76. A gene silencing kit comprising the oligonucleotide of any one of claims 64 to 70 or the dsRNA molecule of any one of claims 1 to 33 or claims 71 to 75.

78. 1. A method for silencing a target gene in a cell, comprising: (i) the double-stranded RNA of any one of claims 1 to 33 or claims 71 to 75, wherein the antisense strand comprises a nucleotide sequence substantially complementary to the target gene; or (ii) the oligonucleotide of any one of claims 64 to 70, comprising a nucleotide sequence substantially complementary to said target gene; into the cell.

79. 1. A method for reducing expression of a target gene in a subject, comprising: (i) the double-stranded RNA of any one of claims 1 to 33 or claims 71 to 75, wherein the antisense strand comprises a nucleotide sequence substantially complementary to the target gene; or (ii) the oligonucleotide of any one of claims 64 to 70, comprising a nucleotide sequence substantially complementary to said target gene; The method comprises administering to the subject either

80. 80. The method of claim 79, wherein the administering is subcutaneous or intravenous.

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