Six membered ring containing oligomers

Abstract

The present disclosure relates generally to six-membered ring containing nucleosides particularly, piperidino nucleosides and oligonucleotides, oligomers derived from the six-membered ring monomers comprising the same.

Description

SIX MEMBERED RING CONTAINING OLIGOMERS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 442,570, filed February 1, 2023, contents of which are incorporated herein by reference in their entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on January 31, 2024, is named “051058-000102WOPT SEQ_list.xml” and is 2,740,425 bytes in size. TECHNICAL FIELD

[0003] The present disclosure relates generally to six-membered nucleosides and oligonucleotides and oligomers comprising the same. BACKGROUND

[0004] There is a need in the art for monomer for modulating oligonucleotide characteristics and / or functionality. The present disclosure addresses some of these needs. SUMMARY

[0005] Despite their recent success in the oligotherapeutic field, phosphorodiamidate morpholinos (PMOs) have several drawbacks. The synthesis of PMOs has difficult to make at scale. The chemical synthesis of PMOs has been conventionally carried out using N-tritylated 5’- chlorophospho-ramidate morpholino monomers which have poor solution-phase stability, long condensation times and low stepwise condensation yields, making PMO synthesis relatively expensive and inaccessible. See, for example, Krishna et al., FEBS Lett. (2019), vol 593(13), pp. 1459-1467. The methods and monomers described herein don’t have these limitations. The methods and monomers described herein allow synthesis of PMOs and similar structures at scale and relatively inexpensively.

[0006] In one aspect, provided herein is an oligonucleotide comprising at least one nucleoside of Formula (IV) (e.g., one):Formula (IV)

[0007] In nucleotides of Fomrula (IV), B’ is an optionally modified nucleobase.

[0008] In nucleotides of Formula (IV), XMcan be CH2, O, NRNor S, where RNis aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars. For example, XMis CH2, O or NH. In some embodiments, XMis CH2. In some embodiments, XMis O. In yet some other embedments, XMis S.

[0009] In nucleotides of Formula (IV), R43can be can be a bond to an intemucleotide linkage to a subsequent nucleotide hydrogen, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or -0-C4-3oalkyl- ON(CH2R8)(CH2R9). In some embodiments of any one the aspects described herein, R43is a bond to an intemucleotide linkage to a subsequent nucleotide. In some other embodiments of any one of the aspects described herein, R43is a soild support or a linker covelantly linked to a solid support. In yet some other embodiments of any one of the aspects described herein, R43is H. In still some other embodiments of any one of the aspects described herein, R43is a nitrogen protecting group, e.g., triphenylmethyl (trityl). In yet some other embodiments of any one of the aspects described herein, R43is hydroxyl or a protected hydroxyl. In yet some other embodiments of any one of the aspects described herein, R43is hydroxyl. In yet some other embodiments of any one of the aspects described herein, R43is a protected hydroxyl.

[0010] In nucleotides of Formula (IV), R45can be a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or 5’-O- C(O)CH2CH2C(O)-) to a solid support hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-l-yl, piperidin-l-yl, or pyrrolidin-l-yl), halogen, alkoxyalkyl (e.g.,2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O- C4-3oalkyl-ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P- 0-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((H0)2(0)P-0-(H0)(0)P-0- P(H0)(0)-0-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha- thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((H0)2(0)P- NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rpis optionally substituted Ci-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(0H)(0)P-0- 5', RP1is alkoxyalkyl, e.g., methoxymethyl (CH20Me) or ethoxymethyl ], (HO)2(X)P-O[-(CH2)a- 0-P(X)(0H)-0]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O- P(X)(0H)-0]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-0- P(X)(0H)-0]b- 5', Me2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H0[-(CH2)a-P(X)(0H)-0]b- 5' , H2N[- (CH2)a-P(X)(0H)-0]b- 5', H[-(CH2)a-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., - CH=CH-XP, Xpis a phosphonate group) or C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), wherein: X is O or S;a and b are each independently 1-10; and each R8and R9is independently H, a targeting ligand (e.g., N-Acetylgalactosamine (GalNAc)), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl.

[0011] In some embodiments, R45is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, phosphate mimic, or a bond to an intemucleotide linkage to a preceding nucleotide, or or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group. For example, R45is hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate, or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group. In some embodiments, R45is a bond to an intemucleotide linkage to a preceding nucleotide. In some embodiments, R45is hydroxyl or protected hydroxyl.

[0012] It is noted only one of R43and R45can be a solid support or a linker covalently bonded to a solid support.

[0013] In some embodiments, the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide. It is noted that the nucleoside of Formula (IV) at 3 ’-end of the oligonucleotide can be linked to the preceding nucleoside by a phosphodiester intemucleotide linkage or a modified intemucleotide. For example, when the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, R45is a bond to a phosphodiester intemucleotide linkage. In another example, when the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, R45is a bond to a phosphorothioate intemucleotide linkage.

[0014] In some embodiments of any one of the aspects described herein, an oligonucleotide described herein comprises two or more consective nucleosides of Formula (IV). For example, the oligonucleotide comprises:where: nDis an interger from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20; or 10-50, or 10-40, or 10-30, or 20-50, or 20-45, or 20-40, or 20-35, or 20-30, or 25-50, or 25- 45, or 25-40, or 25-35, or 25-30);XDis O or S; each RP2is independently optionally substituted Ci-ealkyl (e.g., methyl); andXM, B’, R43and R45are as defined for Formula (IV). Such may be prepared according to methods in the art, including, for example, Kundu et al., J. Org. Chem. 2022, 87, 9466-9478.

[0015] In some embodiments of any one of the aspects described herein, nDis 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. For example, nDis 1, 2, 3, 4, 5 or 6. In some embodiments of any one of the aspects described herien, nDis 1, 2, 3 or 4. For example, nDis 1 or 2.

[0016] In some embodiments of any one of the aspects described herien, XDis O.

[0017] In some oligonucleotides described herein and comprising two or more nucleosides of Formula (IV), all XMare same. For example, all XMare CH2. In some other examples, all XMare O. In some other examples, all XMare S. In some other examples, at least one XMis not O.

[0018] In some embodiments of any one of the aspects described herein, the oliognucleotides is 4 nucleotides in length, nDis 4, and the oligonucleotide does no comprise the sequence 5’-UCAG- 3’.

[0019] In some oligonucleotides described herein and comprising two or more nucleosides of Formula (IV), at least one of XMis CH2 and at least one XMis O.

[0020] In another aspect, provided herein is a compound of Formula (III):(Formula III).

[0021] In compounds of Formula (III), B’ is an optionally modified nucleobase.

[0022] In compounds of Formula (III), XMis CH2, O, NRNor S, where RNis aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars. For example, XMcan be CH2, O or NRN. In some embodiments of any one of the aspects described herein, XMis CH2, O or NH. For example, XMis CH2. In some cases XMis O. In some other non-limiting exampples, XMis S.

[0023] In compounds of Formula (III), R33can be hydrogen, hydroxyl, protected hydroxyl, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxy alkyl (e.g., methoxy ethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), or -0-C4-3oalkyl-ON(CH2R8)(CH2R9). In some embodiments of any one of the aspcets described herein, R33is a nitrogen protecting group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support, a ligand, or a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids. For example, R33is a reactive phosphorous group, a solid support, a linker, a linker covalently bonded to a solid support, or a nitrogen protecting group. In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group, e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)] -thiophosphorami dite. In some other embodiments of any oneof the aspects described herein, R33is a soild support or a linker covelantly linked to a solid support. In yet some other embodiments of any one of the aspects described herein, R33is H. In still some other embodiments of any one of the aspects described herein, R33is a nitrogen protecting group, e.g., truphenylmethyl (trityl).

[0024] In compounds of Formula (III), R35can be a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or 5’-O-C(O)CH2CH2C(O)- ) to a solid support, hydroxy, protected hydroxy, phosphate group, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), -0-C4-3oalkyl- ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)- 0-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P- 0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(0H)(0)P-0-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc...), alkyletherphosphonates (R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (CFbOMe), ethoxymethyl, etc...), (HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a- P(X)(0H)-0]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0- P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-0-P(X)(0H)-0]b- 5', Me2N[- (CH2)a-0-P(X)(0H)-0]b- 5', H0[-(CH2)a-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-P(X)(OH)-O]b- 5', H[- (CH2)a-P(X)(0H)-0]b- 5', Me2N[-(CH2)a-P(X)(OH)-O]b- 5', or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate) group; wherein X is O or S; and a and b are each independently 1- 10); and each R8and R9is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl. In some embodiments of any one of the aspects described herein, R35is hydroxy or protected hydroxy. For example, R35is a protected hydroxyl (e.g., 4,4'-dimethoxytrityl- protected). In yet some other embodiments of any one of the aspects described herein, R35is a phosphate group. In still some other embodiments of any one of the aspects described herein, R35taken together with the carbon to which it is attached can form is a vinyl phosphonate group.

[0025] In compounds of Formula (III), R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group, a C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate) group.

[0026] In some embodiments of any one of the aspects described herein, R35is a reactive phosphorous group. For example, the R35is -OP(O)(RP4)(N(RP2)2), where RP4is Cl and each RP2is methyl.

[0027] It is noted only one of R33and R35can be a reactive phosphorous group, a solid support, or a linker covalently bonded to a solid support.

[0028] In some embodiments of any one of the aspects describd herein, R33is a solid support, or a linker covalently bonded to a solid support. For example, R33is a solid support, or a linker covalently bonded to a solid support, and R35is hydroxyl or protected hydroxyl group. In some compounds of Formula (III), XMis CH2, O, NRNor S; R33is a solid support, or a linker covalently bonded to a solid support; and R35is hydroxyl or protected hydroxyl group (e.g., dimethoxy trityl).

[0029] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group. For example, the R33is -OP(O)(RP4)(N(RP2)2), where RP4is Cl and each RP2is methyl. In some embodiments, R33is a reactive phosphorous group and R35is hydroxyl or protected hydroxyl group. For example, R33is -OP(O)(RP4)(N(RP2)2), where RP4is Cl and each RP2is methyl; and R35is hydroxyl or protected hydroxyl group (e.g., dimethoxytriryl).

[0030] The compounds (III) are useful in the synthesis oligonucleotides. Accordingly, in another aspect, provided herein is an oligonucleotide prepared using a compound of Formula (III). For example, an oligonucleotide comprising nucleoside of Formula (IV).

[0031] In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at one of positions 2-9, counting from the 5 ’end of the oligonucleotide. For example, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at one of positions 2- 8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5 ’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 5, counting from the 5’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 6, counting from the 5 ’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 7, counting from the 5 ’-end of the oligonucleotide. In some embodiments, the oligonucleotide described herein comprises a nucleotide of Formula (IV) at position 8, counting from the 5 ’-end of the oligonucleotide.

[0032] In some embodiments, the oligonucleotide described herein is double-stranded. For example, the oligonucleotide described herein is comprised in a double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantiallycomplementary to the first strand, wherein one of first or second oligonucleotide strand is an oligonucleotide described herein.

[0033] Accordingly, in another aspect, provided herein is a double-stranded nucleic acid comprising a first strand and a second strand complementary to the first strand, and wherein at least one of the first and second strand is an oligonucleotide comprising a nucleotide of Formula (IV) described herein.

[0034] In some embodiments, the double-stranded nucleic acid is a double-stranded RNA. For example, the double-stranded nucleic acid is an siRNA.

[0035] In some embodiments, the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to the sense strand, wherein the antisense strand comprises a nucleotide of Formula (IV). For example, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at one of positions 2-9, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In some embodiments, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5’- end of the antisense strand). In one non-limiting example, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 5, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 6, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In yet another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 7, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In still another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the antisense strand comprises a nucleotide of Formula (IV) at position 8, counting from the 5 ’end of the antisense (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

[0036] In some embodiments, the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to the sense strand, wherein the sense strand comprises a nucleotide of Formula (IV). For example, the double-stranded nucleic acid is an siRNA comprising a sense strand and an antisense strand substantially complementary to thesense strand, wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

[0037] In some embodiments, the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In one non-limiting example, the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In yet another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand). In still another non-limiting example, the double-stranded nucleic acid is an siRNA wherein the sense strand comprises a nucleotide of Formula (IV) at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

[0038] In another aspect, provided herein is a method for inhibiting or reducing the expression of a target gene in a subject. The method comprises administering to the subject: (i) a double- stranded RNA described herein, wherein one of the strands of the dsRNA is complementary to a target gene; and / or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.

[0039] It is noted that in oligonucleotides comprising a nucleotide of Formula (IV), at least one of R43and R45is a bond to a intemucleotide linkage. Thus, when R43is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R45is a bond to an intemucleotide linkageto a preceding nucleotide. Conversely, when R45is not a bond to an intemucleotide linkage to a preceding nucleotide, then R43is a bond to an intemucleotide linkage to a subsequent nucleotide.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing (s) will be provided by the Office upon request and payment of the necessary fee.

[0041] FIG. 1 depicts some exemplary compounds comprising C5 or C4 modified pyrimidines according to some embodiments of the disclosure.

[0042] FIG. 2 depicts some exemplary compounds comprising N2 modified purines according to some embodiments of the disclosure.

[0043] FIG. 3 depicts some exemplary compounds comprising N6 modified purines according to some embodiments of the disclosure.

[0044] FIG. 4 depicts some exemplary compounds comprising N7-deaza or C-8 modified purines according to some embodiments of the disclosure.

[0045] FIG. 5 depicts some exemplary modified car-morpholino, vinylphosphonate and CPG compounds comprising N2 modified purines according to some embodiments of the disclosure.

[0046] FIG. 6 depicts exemplary synthesis scheme for preparing C5-modified pyrimidine car- PMO compounds according to some embodiments of the disclosure.

[0047] FIG. 7 depicts some exemplary ligands.

[0048] FIGS. 8A-8C depict structures of chlorophosphoramidate carbocyclic morpholino monomers (FIG. 8A); PMO, carPMO, and PMO-carPMO chimers (FIG. 8B); and thio-PMO, and PiperazinoPMO chimers (FIG. 8C).

[0049] FIG. 9 depicts crystal structures of selected intermediates.

[0050] FIG. 10 shows overlaid X-ray crystal structures of compound 5, 7, and 8. Atoms are colored teal, pink, and yellow for carbons of 5, 7, and 8, respectively. Oxygen, nitrogen, and silicon are colored gray, blue, and red, respectively.

[0051] FIGS. 11A and 11B are general schemes for synstheis of exemplary car-morpholino and morpholino amidites.

[0052] FIG. 12 are general synthesis schemes for car-morpholino-VP, morpholino-VP- amidite and CPG.

[0053] FIG. 13 show exemplary control sequences with GNA and TNA at Position 7 for off- target mitigation evaluation. Sequences shown from top to bottom are SEQ ID NO: 230 (si-72 sense strand), SEQ ID NO: 231 (si-72 antisense strand), SEQ ID NO: 232 (si-75 sense strand) and SEQ ID NO: 233 (si-75 sense strand).

[0054] FIG. 14 shows structures of some monomer abbreviations used in the nucleic acid sequences described herein.

[0055] FIG. 15A shows exemplary oligonucleotides comprising six membered ring monomers I-IV shown in FIG. 14.

[0056] FIG. 15B shows exemplary oligonucleotides comprising six membered ring monomers Y271-Y274 shown in FIG. 14.

[0057] FIG. 16 shows in vitro results of some exemplary siRNAs targeting mTTR and comprising six membered ring monomers described herein. Sequences shown from top to bottom are SEQ ID NOs: 1 and 2 (control), SEQ ID NOs: 1 and 98 (si-2), SEQ ID NOs: 121 ans 2 (si-25) and SEQ ID NOs: 116 and 2 (si-20).

[0058] FIG. 17 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strand. Sequences shown from top to bottom are SEQ ID NOs: 1 and 2.

[0059] FIG. 18 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.

[0060] FIG. 17 shows in vitro results of some exemplary siRNAs comprising comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.

[0061] FIG. 18 shows in vitro results of some exemplary siRNAs comprising a morpholino monomer in the antisense strand and targeting mTTR and comprising morpholino monomers described herein in the sense strandSequences shown from top to bottom are SEQ ID NOs: 1 and 2.

[0062] FIG. 21 depicts a scheme showing 5’-5-morpholino-U monomer synthesis.

[0063] FIG. 22 depicts a scheme showing 5’-5-morpholino-C monomer synthesis.

[0064] FIG. 23 depicts a scheme showing 5’-5-morpholino-A monomer synthesis.

[0065] FIG. 24 depicts a scheme showing 5’-5-morpholino-G monomer synthesis.

[0066] FIG. 25 depicts a scheme showing 5’-5-morpholino (phosphorami dites) monomers synthesis and oligonucleotide ynthesis strategy.

[0067] FIG. 26 depicts a scheme showing 5’-5-morpholino (chlorophosphorami dates) monomers synthesis and oligonucleotide synthesis strategy.

[0068] FIG. 27 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis.

[0069] FIG. 28 depicts a scheme showing A-acetyl piperazino (phosphoramidites) monomers synthesis.

[0070] FIG. 29 depicts a scheme showing A-acetyl piperazino (chlorophosphoramidates) monomers synthesis and oligonucleotide synthesis scheme.

[0071] FIG. 30 depicts a scheme showing N- acetyl piperazino (phosphoramidites) monomers synthesis and oligonucleotide synthesis scheme.

[0072] FIG. 31 depicts some exemplary modified PMO sequences. Seqeunces shown are, from top to bottom, SEQ ID NOs: 241-244.DETAILED DESCRIPTION

[0073] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.

[0074] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited 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 any purpose.Nucleobase

[0051] In some embodiments of the various aspects described herein, B’ is an optionally modified nucleobase. It is noted that the nucleobase can be a natural or non-natural nucleobase. By a “non-natural nucleobase” is meant a nucleobase other than adenine, guanine, cytosine, uracil, or thymine. Exemplary non-natural nucleobases include, but are not limited to, 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 cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo,amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5 -trifluoromethyl and other 5 -substituted uracils and cytosines, 7-methylguanine, 5 -substituted pyrimidines, 6- azapyrimi dines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil (e.g., 5 -methyluracil), 5 -alkylcytosines (e.g., 5-methylcytosine), 7-alkylguanine, 5- alkyl cytosine, 7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3 -methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5 -nitroindole, 3 -nitropyrrole, 5- methoxyuracil, uracil-5-oxyacetic acid, 5 -methoxy carbonylmethyluracil, 5-methyl-2-thiouracil, 5- methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino- 3carboxypropyl)uracil, 3 -methylcytosine, 5-methylcytosine, N4-acetyl cytosine, 2-thiocytosine, N6-methyladenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N- methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia of Polymer Science and Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, content of all which is incorporated herein by reference.

[0052] In some embodiments, the non-natural nucleobase can be selected from the group consisting of inosine, xanthine, hypoxanthine, nubularine, isoguanisine, tubercidine, 2- (halo)adenine, 2-(alkyl)adenine, 2-(propyl)adenine, 2-(amino)adenine, 2-(aminoalkyll)adenine,2-(aminopropyl)adenine, 2-(methylthio)-N6-(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, N6-(isopentyl)adenine, N6-(methyl)adenine, N6, N6-(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, 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-(propynyl)cytosine, 5-(trifluoromethyl)cytosine, 6-(azo)cytosine, N4-(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-(l,3- diazole-l-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-(methoxycarbonyl-methyl)uracil, 5-(propynyl)uracil, 5-(propynyl)uracil, 5-(trifluoromethyl)uracil, 6-(azo)uracil, dihydrouracil, N3-(methyl)uracil, 5-uracil (i.e., pseudouracil), 2-(thio)pseudouracil,4-(thio)pseudouracil,2,4- (dithio)psuedouracil,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, 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 -(aminocarbonyl ethylenyl)- 4-(thio)pseudouracil, l-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil,1 -(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1 -(aminoalkylamino-carbonylethylenyl)- 2(thio)-pseudouracil, 1 -(aminoalkylaminocarbonyl ethylenyl)-4-(thio)pseudouracil, l-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, l,3-(diaza)-2-(oxo)-phenoxazin- 1-yl, l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, l-(aza)-2- (thio)-3-(aza)-phenthiazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl, 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-substituted l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7- substituted l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl, 7-(aminoalkylhy droxyl)-!, 3 -(diaza)-2- (oxo)-phenoxazin- 1 -yl, 7-(aminoalkylhy droxyl)- 1 -(aza)-2-(thio)-3 -(aza)-phenoxazin- 1 -yl, 7- (aminoalkylhydroxyl)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl, 7-(aminoalkylhy droxyl)- l-(aza)-2- (thio)-3 -(aza)-phenthiazin- 1 -yl, 7-(guanidiniumalkylhy droxyl)- 1 ,3 -(diaza)-2-(oxo)-phenoxazin- 1 - yl, 7-(guanidiniumalkylhydroxyl)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl, 7-(guanidiniumalkyl- hy droxyl)- 1 ,3 -(diaza)-2-(oxo)-phenthiazin- 1 -yl, 7-(guanidiniumalkylhy droxyl)- 1 -(aza)-2-(thio)- 3-(aza)-phenthiazin-l-yl, l,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine, hypoxanthine, nubularine, tubercidine, 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, pyrrolopyrizinyl, isocarbostyrilyl, 7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl, 2,4,5- (trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl, phenyl, napthalenyl, 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, N2-substituted purines, N6-substituted purines, ( / -substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[0053] In some embodiments, a non-natural nucleobase is a modified nucleobase, i.e., the nucleobase comprises a nucleobase modification described herein, e.g., the nucleobase is a substituted or modified analog of any of the natural nucleobases. Examples of the nucleobase modifications include, but not limited to: C-5 pyrimidine with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities, N2- and N6- with an alkyl group or aminoalkyls and other cationic groups such as guanidinium and amidine functionalities of purines, G-clamps, guanidinium G-clamps, and pseudouridine known in the art.

[0054] In some embodiments of any one of the aspects, the non-natural nucleobase is a universal nucleobase. As used herein, a universal nucleobase is any modified or unmodified natural or non-natural nucleobase that can base pair with all of adenine, cytosine, guanine and uracil without substantially affecting the melting behavior, recognition by intracellular enzymes or activity of the oligonucleotide comprising the universal nucleobase. Some exemplary universal nucleobases include, but are not limited to, 2,4-difluorotoluene, nitropyrrolyl, nitroindolyl, 8-aza- 7-deazaadenine, 4-fluoro-6-methylbenzimidazle, 4-methylbenzimidazle, 3 -methyl isocarbostyrilyl, 5- methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6- methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4, 5 -trimethylphenyl, 4- methylinolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenyl, tetracenyl, pentacenyl, and structural derivatives thereof.

[0055] In some embodiments of any one of the aspects described herein, the non-matural nucleobase is a protected nucleobase. As used herein, a “protected nucleobase” referes to a nucleobase comprising a nitrogen protecting group, and / or an oxygen protecting group, and / or a sulfur protecting group.

[0056] In some embodiments of any one of the aspects described herein, the non-natural nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil.

[0057] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C4 position.

[0058] In some embodiments of any one of the aspects described herein, the nucleobase is a pyrimidine modified at the C5 position.

[0059] 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.

[0060] In some embodiments of any one of the aspects described herein, the nucleobase is a purine modified at the C6 position.

[0061] In some embodiments of any one of the aspects described herein, the nucleobase is a N-7 deaza purine, optionally modified at the N7 position.

[0062] In some embodiments of any one of the aspects described herein, the nucleobase is selected from the group consistingindependently liphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.

[0063] In some embodiments of any one of the aspects described herein, XMcan be CH2, O,S, or NRN.

[0064] In some embodiments of any one of the aspects described herein, XMis CH2.

[0065] In some embodiments of any one of the aspects described herein, XMis O.

[0066] In some embodiments of any one of the aspects described herein, XMis S.

[0067] In some embodiments of any one of the aspects described herein, XMis NRN. For example, XMis NH.R33

[0068] In some embodiments of any one of the aspects described herein, R33can be hydrogen, hydroxyl, protected hydroxyl, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or -0-C4-3oalkyl- ON(CH2R8)(CH2R9).

[0069] In some embodiments of any one of the aspects described herein, R33is a nitrogen protecting group. For example, R33' can be triphenylmethylamine (Tr), [(4- methoxyphenyl)diphenylmethyl]amine (MMTr), 4,4'-dimethoxytriphenylmethyl (DMTr) ortrifluoroacetamide. In some embodiments of any one of the aspects described herein, R33is triphenylmethylamine or trifluoroacetamide. For example, R33is triphenylmethylamine.

[0070] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorus group. For example, R33is -OP(ORP)(N(RP2)2), -OP(SRP)(N(RP2)2), - OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(NRP2)2, -OP(O)(ORP)H, - OP(S)(ORP)H, -OP(O)(SRP)H, -OP(O)(ORP)RP3, -OP(S)(ORP)RP3, or -OP(O)(SRP)RP3.

[0071] In some embodiments of any one of the aspects, R33is -OP(ORP)(N(RP2)2), - OP(SRP)(N(RP2)2), -OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), - OP(O)(ORP)H, -OP(S)(ORP)H, where RPis an optionally substituted C1-6alkyl, each RP2is independently optionally substituted C1-6alkyl; and each RP3is independently optionally substituted C1-6alkyl.

[0072] In some embodiments of any one of the aspects, R33is -OP(ORP)(N(RP2)2). For example, the R33is -OP(ORP)(N(RP2)2), where RPis cyanoethyl (-CH2CH2CN) and each RP2is isopropyl.

[0073] In some embodiments of any one of the aspects descried herein, R33is a solid support or a linker covalently attached to a solid support. For example, R33is –OC(O)CH2CH2C(O)NH-Z, where Z is a solid support.

[0074] In some embodiments of any one of the aspects described herein, R33is - O(CH2CH2O)rCH2CH2OR334, where r can be 1-50; R334is independently for each occurrence H, C1-C30alkyl, cyclyl, heterocyclyl, aryl, heteroaryl, aralkyl, sugar or R335; and R335is independently for each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.

[0075] In some embodiments of any one of the aspects described herein, R33is - (CH2CH2NH)sCH2CH2-R335, where s can be 1-50 and R335can be independently for each occurrence amino (NH2), alkylamino, dialkylamino, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino.

[0076] In some embodiments of any one of the aspects described herein, R33is hydrogen.

[0077] In some embodiments of any one of the aspects described herein, R33is hydroxyl or protected hydroxyl. In some embodiments of any one of the aspects described herein, R33is hydroxyl. In some embodiments of any one of the aspects described herein, R33is protected hydroxyl.

[0078] In some embodiments of any one of the aspects described herein, R33is C1-C30alkoxy optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R33is C1-C30alkoxy optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy. In some embodiments of any one of the aspects described herein, R33is –O(CH2)tCH3, where t is 1-21. For example, t is 14, 15, 16, 17 or 18. In one non-limiting example, t is 16.

[0079] In some embodiments of any one of the aspects, R33is –O(CH2)uR337, where u is 2-10; R337is C1-C6alkoxy, amino (NH2), CO2H, OH or halo. For example, R337is -CH3or NH2. Accordingly, in some embodiments of any one of the aspects described herein, R33is –O(CH2)u- OMe or R33is –O(CH2)uNH2. For example, u is 2, 3, 4, 5 or 6. In some embodiments, u is 2, 3 or 6. In one non-limiting example, u is 2. In another non-limiting example, u is 3 or 6.

[0080] In some embodiments of any one of the aspects described herein, R33is a C1- C6haloalkyl. For example, R33is a C1-C4haloalkyl. In some embodiments of any one of the aspects described herein, R33is –CF3, -CF2CF3, -CF2CF2CF3 or -CF2(CF3)2.

[0081] In some embodiments of any one of the aspects described herein, R33is – OCH(CH2OR338)CH2OR339,where R338and R339independently are H, optionally substituted C1- C30alkyl, optionally substituted C2-C30alkenyl or optionally substituted C2-C30alkynyl. For example, R338and R339independently are optionally substituted C1-C30alkyl.

[0082] In some embodiments of any one of the aspects described herein, R33is – CH2C(O)NHR3310, where R3310is H, optionally substituted C1-C30alkyl, optionally substituted C2- C30alkenyl or optionally substituted C2-C30alkynyl. For example, R3310is H or optionally substituted C1-C30alkyl. In some embodiments, R3310is optionally substituted C1-C6alkyl.

[0083] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R33is a reactive phosphorous or a linker covalently attached to a solid support.

[0084] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite). R35

[0085] In compounds of Formula (III), R35can be hydroxy, protected hydroxy, phosphate group, optionally substituted C1-30alkyl, optionally substituted C2-30alkenyl, optionally substitutedC2-30alkynyl, optionally substituted C1-30alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl- ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.

[0086] In some embodiments of the various aspects described herein, R35is R551, optionally substituted C1-6alkyl-R551, optionally substituted -C2-6alkenyl-R551, or optionally substituted -C2-6alkynyl-R551, where R551can be –OR552, -SR553, hydrogen, a phosphorous group, a solid support or a linker to a solid support. When R551is –OR552, R552can be H or a hydroxyl protecting group. Similarly, when R551is –SR553, R553can be H or a sulfur protecting group.

[0087] In some embodiments of any one of the aspects described herein, R35is –OR552or - SR553.

[0088] In some embodiments of any one of the aspects described herein, R552is a hydroxyl protecting group. Exemplary hydroxyl protecting groups for R552include, but are not limited to, benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4′-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9- yl (MOX). In some embodiments of any one of the aspects described herein, R35is –OR552and R552is 4,4′-dimethoxytrityl (DMT), e.g., R35is –O-DMT.

[0089] In some embodiments of any one of the aspects described herein, R552is a phosphate group, e.g., R552is dimethylaminochlorophosphate (-P(O)(NMe2)Cl).

[0090] In some embodiments of any one of the aspects described herein, the methylene connecting the R35to the rest of the compound of Formula (III) is absent and R35is connected directly to the rest of the compound of Formula (III).

[0091] In some embodiments of any one of the aspects described herein, R35is –CH(R554)- R551, where R554is hydrogen, halogen, optionally substituted C1-C30alkyl, optionally substituted C2-C30alkenyl, optionally substituted C2-C30alkynyl, or optionally substituted C1-C30alkoxy.

[0092] In some embodiments of any one of the aspects, when R35is –CH(R554)-R551, R554is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R554isH. In some other non-limiting examples, R554is C1-C30alkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy.

[0093] In some embodiments of the various aspects described herein, R35is –CH(R554)-O-R552, where R554is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R554is H. In some other non-limiting examples, R554is C1-C30alkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy.

[0094] In some embodiments of the various aspects described herein, R35is optionally substituted C1-6alkyl-R551or optionally substituted -C2-6alkenyl-R551,

[0095] In some embodiments of any one of the aspects described herein, R35is – C(R554)=CHR551. It is noted that the double bond in –C(R554)=CHR551can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, Rdis – C(R554)=CHR551and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, Rdis –C(R554)=CHR551and wherein the double bond is in the trans configuration.

[0096] In some embodiments of any one of the aspects described herein, R35is –CH=CHR551.

[0097] In some embodiments of any one of the aspects, when R35is –C(R554)=CHR551, R554is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R551is a phosphorous group. For example, R35is –CH=CHR551.

[0098] In some embodiments of any one of the aspects described herein, R551is a reactive phosphorous group.

[0099] In some embodiments of any one of the aspects, R35is -CH=CH-P(O)(OR555)2, - CH=CH-P(S)(OR555)2, -CH=CH-P(S)(SR556)(OR555), -CH=CH-P(S)(SR556)2, -CH=CH- OP(O)(OR555)2, -CH=CH-OP(S)(OR555)2, -CH=CH-OP(S)(SR556)(OR555), -CH=CH-OP(S)(SR556)2, -CH=CH-SP(O)(OR555)2, -CH=CH-SP(S)(OR555)2, -CH=CH-SP(S)(SR556)(OR555), or -CH=CH -SP(S)(SR556)2, where each R555is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group; and each R556is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or a sulfur-protecting group.

[0100] In some embodiments of any one of the aspects, at least one R555in -P(O)(OR555)2, - P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555) is hydrogen.

[0101] In some other embodiments of any one of the aspects, at least one R555in -P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, or -SP(S)(SR556)(OR555) is not hydrogen. For example, at least one at least one R555in P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.

[0102] In some embodiments of any one of the aspects, at least one R555is H and at least one R555is other than H in -P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), SP(O)(OR555)2, -SP(S)(OR555)2, and -SP(S)(SR556)(OR555).

[0103] In some embodiments of any one of the aspects, all R555are H in -P(O)(OR555)2, - P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), - OP(S)(SR556)2, -SP(O)(OR555)2, -SP(S)(OR555)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.

[0104] In some embodiments of any one of the aspects, all R555are other than H in in - P(O)(OR555)2, -P(S)(OR555)2, -P(S)(SR556)(OR555), -OP(O)(OR555)2, -OP(S)(OR555)2, - OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(O)(OR555)2, -SP(S)(OR555)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.

[0105] In some embodiments of any one of the aspects, at least one R556in - P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2is H.

[0106] In some embodiments of any one of the aspects, at least one R556in - P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2 is other than H. For example, at least one R556in -P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, - SP(S)(SR556)(OR555), and -SP(S)(SR556)2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.

[0107] In some embodiments of any one of the aspects, at least one R556is H and at least one R556is other than H in -P(S)(SR556)2, -OP(S)(SR556)2and -SP(S)(SR556)2.

[0108] In some embodiments, all R556are H in -P(S)(SR556)(OR555), -P(S)(SR556)2, - OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.

[0109] In some embodiments, all R556are other than H in -P(S)(SR556)(OR555), -P(S)(SR556)2, -OP(S)(OR555)2, -OP(S)(SR556)(OR555), -OP(S)(SR556)2, -SP(S)(SR556)(OR555), and -SP(S)(SR556)2.

[0110] In some embodiments of any one of the aspects, R35is -CH=CH-P(O)(OR555)2, where each R555is H or an oxygen protecting group.

[0111] In some embodiments of any one of the aspects, R35is hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate or phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group. For example, R35is hydroxyl, protected hydroxyl, cyclopropylphosphonate, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, or a phosphate mimic. In other examples, R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group.

[0112] In some embodiments of any one of the aspects described herein, R35is a monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma- thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic; or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0113] In some embodiments of any one of the aspects described herein, R35is a reactive phosphorus group. For example, R33is -OP(ORP)(N(RP2)2), -OP(SRP)(N(RP2)2), - OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), -OP(O)(ORP)H, - OP(S)(ORp)H, -OP(O)(SRp)H, -OP(O)(ORP)RP3, -OP(S)(ORP)RP3, -OP(O)(SRP)RP3, - OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2).

[0114] In some embodiments of any one of the aspects, R35is -OP(ORP)(N(RP2)2), - OP(SRP)(N(RP2)2), -OP(O)(ORP)(N(RP2)2), -OP(S)(ORP)(N(RP2)2), -OP(O)(SRP)(N(RP2)2), - OP(O)(ORP)H, -OP(S)(ORP)H, -OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2), where each RPis independently an optionally substituted C1-6alkyl, each RP2is independently optionally substituted C1-6alkyl; and each RP3is independently optionally substituted C1-6alkyl. For example, R35is OP(O)(RP3)(N(RP4)2), or -OP(S)(RP4)(N(RP2)2).

[0115] In some embodiments of any one of the aspects, R35is –OP(O)(RP4)(N(RP2)2). For example, the R35is –OP(O)(RP4)(N(RP2)2), where RP4is Cl and each RP2is methyl.

[0116] In some embodiments of any one of the aspects descried herein, R35is a solid support or a linker covalently attached to a solid support. For example, R35is –OC(O)CH2CH2C(O)NH-Z, where Z is a solid support.

[0117] In some embodiments of any one of the aspects described herein, R33is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R35is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, R33is H or a nitrogen protecting group (e.g., trityl); and R35is a reactive phosphorous group (e.g., –OP(O)(RP4)(N(RP2)2). In some embodiment, R33is a nitrogen protecting group (e.g., trityl); and R35is –OP(O)(RP4)(N(RP2)2), where RP4is halogene (e.g., Cl) and each RP2is independently C1-C6alkyl, e.g., each RP2is independently methyl.

[0118] In some embodiments of any one of the aspects described herein, XMis O, S or CH2; R33is H, hydroxyl, protected hydroxyl, nitrogen protecting group, a linker, a ligand or a ligand covalently attached to one or more ligands; and R35is reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support. For example, XMis CH2; R33is H or a nitrogen protecting group (e.g., trityl); and R35is a reactive phosphorous group (e.g., – OP(O)(RP4)(N(RP2)2). In some embodiment, XMis CH2; R33is a nitrogen protecting group (e.g., trityl); and R35is –OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently C1-C6alkyl, e.g., methyl.

[0119] In other examples, XMis CH2; R33is hydroxyl or protected hydroxyl; and R35is a reactive phosphorous group (e.g., –OP(O)(RP4)(N(RP2)2). In some embodiment, XMis CH2; R33is protected hydroxyl; and R35is –OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently C1-C6alkyl, e.g., methyl.

[0120] In some embodiments of any one of the aspects described herein, XMis O; R33is H or a nitrogen protecting group (e.g., trityl); and R35is a reactive phosphorous group (e.g., – OP(O)(RP4)(N(RP2)2). For example, XMis O; R33is a nitrogen protecting group (e.g., trityl); andR35is -OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently Ci- Cealkyl, e.g., methyl.

[0121] In some embodiments of any one of the aspects described herein, XMis O; R33is hydroxyl or protected hydroxyl and R35is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XMis O; R33is a nitrogen protecting group (e.g., trityl); and R35is -OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently Ci- Cealkyl, e.g., methyl.

[0122] In some embodiments of any one of the aspects described herein, XMis S; R33is H or a nitrogen protecting group (e.g., trityl); and R35is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XMis S; R33is a nitrogen protecting group (e.g., trityl); and R35is -OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently Ci- Cealkyl, e.g., methyl.

[0123] In some embodiments of any one of the aspects described herein, XMis S; R33is hydroxyl or protected hydroxyl; and R35is a reactive phosphorous group (e.g., - OP(O)(RP4)(N(RP2)2). For example, XMis S; R33is a nitrogen protecting group (e.g., trityl); and R35is -OP(O)(RP4)(N(RP2)2), where RP4is halogen (e.g., Cl) and each RP2is independently Ci- Cealkyl, e.g., methyl.

[0124] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0125] . For example, R33is a reactive phosphorous or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma- thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0126] In some embodiments of any one of the aspects described herein, R33is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B-thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0127] In some embodiments of any one of the aspects described herein, R33is a solid support, a linker or a linker covalently attached to a solid support; R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0128] In some embodiments of any one of the aspects described herein, XMis CEE; R33is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XMis CEE; R33is a reactive phosphorous or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0129] In some embodiments of any one of the aspects described herein, XMis CEE; R33is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrohdinyl)]-thiophosphoramidite); and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e (e.g., E or Z vinylphosphonate) group.

[0130] In some embodiments of any one of the aspects described herein, XMis CEE; R33is a solid support, a linker or a linker covalently attached to a solid support; R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0131] In some embodiments of any one of the aspects described herein, XMis O; R33is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solidsupport; and R35is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XMis O; R33is a reactive phosphorous or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl (e.g., 4,4'- dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0132] In some embodiments of any one of the aspects described herein, XMis O; R33is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0133] In some embodiments of any one of the aspects described herein, XMis O; R33is a solid support, a linker or a linker covalently attached to a solid support; R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0134] In some embodiments of any one of the aspects described herein, XMis S; R33is a reactive phosphorous group, a solid support, a linker, or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates, alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group. For example, XMis S; R33is a reactive phosphorous or a linker covalently attached to a solid support; and R35is hydroxyl, protected hydroxyl (e.g., 4,4'- dimethoxytrityl-protected), monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, alkylphosphonates,alkyletherphosphonates, dialkyl terminal phosphates, or a phosphate mimic, or R35taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0135] In some embodiments of any one of the aspects described herein, XMis S; R33is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- diisopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, or 3'-[(B- thiobenzoylethyl)-(l-pyrrolidinyl)]-thiophosphoramidite); and R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.

[0136] In some embodiments of any one of the aspects described herein, XMis S; R33is a solid support, a linker or a linker covalently attached to a solid support; R35is hydroxyl, protected hydroxyl (e.g., 4,4'-dimethoxytrityl-protected) or or R35taken together with the carbon to which it is attached can form a vinylphosphonate (e.g., E or Z vinylphosphonate) group.R43

[0137] In some embodiments of any one of the aspects described herein, R43can be a bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group.

[0138] In some embodiments of any one of the aspects described herein, R43is bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, or a nitrogen protecting group.

[0139] In some embodiments of any one of the aspects described herein, R43is a bond to an intemucleotide linkage to a subsequent nucleotide.

[0140] In some embodiments of any one of the aspects described herein, R43is a solid support, or a linker (e.g., -C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) covalently bonded to a solid support.

[0141] In some embodiments of any one of the aspects described herein, R43is hydrogen or a nitrogen protecting group.

[0142] In some embodiments of any one of the aspects described herein, R43is hydroxy or protected hydroxyl.

[0143] In some embodiments of any one of the aspects described herein R43is – P(XD)(N(RP2)2)-R43’, where XDis O or S; each RP2is independently optionally substituted C1-6alkyl (e.g., methyl); and R43’is a bond to a subsequent nucleoside, e.g., a bond to 5’ oxygen of a subsequent nucleoside. For example, R43is –P(O)(N(CH3)2)-R43’. R45

[0144] In some embodiments of any one of the aspects described herein, R45can be a bond to an internucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded a solid support, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), -O-C4-30alkyl- ON(CH2R8)(CH2R9), , monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)- O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P- O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…), (HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a- P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[- (CH2)a-O-P(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 a and b are each independently 1-10); or R45taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0145] In some embodiments of any one of the aspects described herein, R45can be a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate (phosphorodithioate), phosphorothiolate, alpha-thiotriphosphate, beta- thiotriphosphate, gamma-thiotriphosphate, phosphoramidates, or alkylphosphonates; or R45takentogether with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.

[0146] In some embodiments of any one of the aspects described herein, R45is a bond to an internucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C2-30alkenyl, or optionally substituted C1-30alkoxy; or R45taken together with the carbon to which it is attached can form a vinylphosphonate (VP) group or a cyclopropylphosphonate group.In some embodiments of any one of the aspects described herein, R45is a bond to an internucleotide linkage to a preceding nucleotide.

[0147] In some embodiments of any one of the aspects described herein, R45is a hydroxyl or protected hydroxyl.

[0148] In some embodiments of any one of the aspects described herein, R45is optionally substituted C2-30alkenyl or optionally substituted C1-30alkoxy.

[0149] In some embodiments of any one of the aspects described herein, R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

[0150] In some embodiments of any one of the aspects described herein, the methylene connecting the R45to the rest of the nucleoside of Formula (VI) is absent and R45is connected directly to the rest of the nucleoside of Formula (IV).

[0151] In some embodiments of any one of the aspects descried herein, R45is –CH(R451)-X5- R452, where X5is absent, a bond or O; R451is hydrogen, optionally substituted C1-30alkyl, optionally substituted -C2-30alkenyl, or optionally substituted -C2-30alkynyl, and R452is a bond to an internucleoside linkage to the preceding nucleotide.

[0152] In some embodiments of any one of the aspects described herein, X5is O or a bond. For example, X5is O. In some other embodiments of any one of the aspects described herein, X5is absent, i.e., R45is–CH(R451)R452.

[0153] In some embodiments of the various aspects described herein, R45is –CH(R451)-R452or –C(R451)=CHR452, where R451is hydrogen, optionally substituted C1-30alkyl, optionally substituted -C2-30alkenyl, or optionally substituted -C2-30alkynyl, and R452is a bond to an internucleoside linkage to the preceding nucleotide.

[0154] In some embodiments of the various aspects described herein, R45is –CH(R451)-X5- R452. For example, R45is –CH(R451)-X5-R452and where R451is H or C1-C30alkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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(0)- alkyl, C(0)- alkyl, alkylcarbonylaminyl, CH2 — [CH(OH)]m— (CH2)P— OH, CH2— [CH(OH)]m— (CH2)P— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R451is H. In some other non-limiting examples, R451is Ci-Csoalkyl optionally substituted with a NH2, OH, C(0)NH2, COOH, halo, SH, or Ci-Cealkoxy.

[0155] In some embodiments of the various aspects described herein, R45is -CH(R451)-O- R452, where R451is H or Ci-Csoalkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(O)NH2, COOH, COOMe, acetyl, (Ci-Cs)alkyl, O(Ci-Cs)alkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)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— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. For example, R451is H. In some other non-limiting examples, R451is Ci-Csoalkyl optionally substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or Ci-Cealkoxy.

[0156] In some embodiments of any one of the aspects described herein, R45is - C(R451)=CHR452. It is noted that the double bond in -C(R451)=CHR452can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, R45is - C(R451)=CHR452and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, R45is is -C(R451)=CHR452and wherein the double bond is in the trans configuration. In some embodiments of any one of the aspects described herein, R45is -CH=CHR452.

[0157] In some embodiments of any one of the aspects described herein, R452is a bond to an intemucleoside linkage to the preceding nucleotide.

[0158] In embodiments of the various aspects described herein, R45is optionally substituted Ci-ealkyl-R453, optionally substituted -C2-6alkenyl-R453, or optionally substituted -C2-6alkynyl-R453. In embodiments of the various aspects described herein, R453can be -OR454, -SR455, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or - SP(S)(SR457)2; where R454is hydrogen or oxygen protecting group; R455is hydrogen or sulfur protecting group; each R456is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an oxygen-protecting group; andeach R457is independently hydrogen, optionally substituted Ci-3oalkyl, optionally substituted C2- soalkenyl, or optionally substituted C2-3oalkynyl, or a sulfur-protecting group.

[0159] In some embodiments of any one of the aspects, at least one R456in -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456) is hydrogen.

[0160] In some other embodiments of any one of the aspects, at least one R456in -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, or -SP(S)(SR457)(OR456) is not hydrogen. For example, at least one at least one R456in P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456) is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2- soalkynyl, or an oxygen-protecting group.

[0161] In some embodiments of any one of the aspects, at least one R456is H and at least one R456is other than H in -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), SP(O)(OR456)2, -SP(S)(OR456)2, and -SP(S)(SR457)(OR456).

[0162] In some embodiments of any one of the aspects, all R456are H in -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), - OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.

[0163] In some embodiments of any one of the aspects, all R456are other than H in in - P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.

[0164] In some embodiments of any one of the aspects, at least one R457in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2is H.

[0165] In some embodiments of any one of the aspects, at least one R457in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2 is other than H. For example, at least one R457in - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(S)(SR457)(OR456), and -SP(S)(SR457)2 is optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl, or an sulfur-protecting group.

[0166] In some embodiments of any one of the aspects, at least one R457is H and at least one R457IS other than H in -P(S)(SR457)2, -OP(S)(SR457)2and -SP(S)(SR457)2.

[0167] In some embodiments, all R457are H in -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.

[0168] In some embodiments, all R457are other than H in -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(S)(SR457)(OR456), and -SP(S)(SR457)2.

[0169] In some embodiments of any one of the aspects described herein, R45is optionally substituted -C2-6alkenyl-R453. For example, R45is -C2-6alkenyl-R453, where C2-6alkenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R453is -P(O)(OR456)2, -P(S)(OR456)2, - P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), - OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or -SP(S)(SR457)2.

[0170] In some embodiments of any one of the aspects, R45is –CH=CHR453. It is noted that a double bond in the optionally substituted -C2-6alkenyl-R453can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects, R45is –CH=CHR453and wherein the double bond is in the cis configuration. In some other embodiments of any one of the aspects, R45is –CH=CHR453and wherein the double bond is in the trans configuration.

[0171] In some embodiments of any one of the aspects, R45is –CH=CH-P(O)(OR456)2, – CH=CH-P(S)(OR456)2, –CH=CH-P(S)(SR457)(OR456), –CH=CH-P(S)(SR457)2, –CH=CH- OP(O)(OR456)2, –CH=CH-OP(S)(OR456)2, –CH=CH-OP(S)(SR457)(OR456), –CH=CH- OP(S)(SR457)2, –CH=CH-SP(O)(OR456)2, –CH=CH-SP(S)(OR456)2, –CH=CH- SP(S)(SR457)(OR456), or –CH=CH -SP(S)(SR457)2. For example, R45is –CH=CH-P(O)(OR456)2.

[0172] In some embodiments, of any one of the aspects, R454is hydrogen or an oxygen protecting group. For example, R454is hydrogen or 4,4′-dimethoxytrityl (DMT). In some preferred embodiments, R454is H.

[0173] In some embodiments of any one of the aspects described herein, R45is optionally substituted –C1-6alkenyl-R453. For example, R45is –C1-6alkenyl-R453, where C1-6alkenyl is optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6; and R453is -OR454, -SR455, -P(O)(OR456)2, - P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, - OP(S)(SR457)(OR456), -OP(S)(SR457)2, -SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or - SP(S)(SR457)2.

[0174] In some embodiments of any one of the aspects described herein, R45can be -CH(R458)- R453, where R453is -OR454, -SR455, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), - P(S)(SR457)2, -OP(O)(OR456)2, -OP(S)(OR456)2, -OP(S)(SR457)(OR456), -OP(S)(SR457)2, - SP(O)(OR456)2, -SP(S)(OR456)2, -SP(S)(SR457)(OR456), or -SP(S)(SR457)2; and R458is H, optionally substituted Ci-3oalkyl, optionally substituted C2-3oalkenyl, or optionally substituted C2-3oalkynyl.

[0175] In some embodiments of any one of the aspects described herein, R458is H or Ci- Csoalkyl optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(0)NH2, COOH, COOMe, acetyl, (Ci- Cs)alkyl, O(Ci-Cs)alkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)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(0H)]m— (CH2)P— OH, CH2— [CH(0H)]m— (CH2)P— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6. In one non-limiting example, R458is H. In some other non-limiting examples, R458is Ci-Csoalkyl optionally substituted with a substituent selected from NH2, OH, C(O)NH2, COOH, halo, SH, and Ci-Cealkoxy.

[0176] In some embodiments of any one of the aspects described herein, R45is -CH(R458)-O- R459, where R459is H, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(O)(OR456)2. For example, R45is -CH(R458)-O-R459, where R458is H or optionally substituted Ci-C3oalkyl and R459is H or -P(O)(OR456)2.

[0177] In some embodiments of any one of the aspects described herein, R45is -CH(R458)-S- R60, where R60is H, -P(O)(OR456)2, -P(S)(OR456)2, -P(S)(SR457)(OR456), -P(S)(SR457)2, - OP(O)(OR456)2.Ligands

[0166] Without wishing to be bound by a theory, ligands modify one or more properties of the attached molecule (e.g., the oligonucleotide described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Ligands are routinely used in the chemical arts and are linked directly or via an optional linking moiety or linking group to a parent compound. A preferred list of ligandsincludes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes.

[0167] Preferred ligands amenable to the present invention include lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053); a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765); a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533); an aliphatic chain, e.g., dodecandiol 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); a phospholipid, e.g., di-hexadecyl-rac-glycerol or tri ethyl ammonium- 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); a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969); adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651); apalmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229); or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277, 923).

[0168] Ligands can include naturally occurring molecules, or recombinant or synthetic molecules. Exemplary ligands include, but are not limited to, polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxylpropyl)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-ethylacryllic acid), N-isopropylacrylamide polymers, polyphosphazine, polyethylenimine, cationic groups, spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, mucin, glycosylated polyaminoacids, transferrin, bisphosphonate, polyglutamate, poly aspartate, aptamer, asialofetuin, hyaluronan, procollagen, immunoglobulins (e.g., antibodies), insulin, transferrin, albumin, sugar-albumin conjugates, intercalating agents (e.g., acridines), cross- linkers (e.g. psoralen, mitomycin 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, adamantane acetic acid, 1- pyrene butyric 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)cholenic acid, dimethoxytrityl, or phenoxazine), peptides (e.g., an alpha helical peptide, amphipathic peptide, RGD peptide, cell permeation peptide, endosomolytic / fusogenic peptide), alkylating agents, phosphate, amino, mercapto, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport / absorption facilitators (e.g., naproxen, aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, AP, antibodies, hormones and hormone receptors, lectins, carbohydrates, multivalent carbohydrates, vitamins (e.g., vitamin A, vitamin E, vitamin K, vitamin B, e.g., folic acid, B12, riboflavin, biotin and pyridoxal), vitamin cofactors, lipopolysaccharide, an activator of p38 MAP kinase, an activator of NF-KB, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, myoservin, tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, gamma interferon, natural or recombinant low density lipoprotein (LDL), natural or recombinant high- density lipoprotein (HDL), and a cell-permeation agent (e.g., a.helical cell-permeation agent).

[0169] Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; a, 0, or y peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic ligand can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0170] Exemplary amphipathic peptides include, but are not limited to, cecropins, ly cotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H2A peptides, Xenopus peptides, esculentinis-1, and caerins.

[0171] As used herein, the term “endosomolytic ligand” refers to molecules having endosomolytic properties. Endosomolytic ligands promote the lysis of and / or transport of the composition of the invention, or its components, from the cellular compartments such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies within the cell, to the cytoplasm of the cell. Some exemplary endosomolytic ligands include, but are not limited to, imidazoles, poly or oligoimidazoles, linear or branched polyethyleneimines (PEIs), linear and brached poly amines, e.g. spermine, cationic linear and branched polyamines, polycarboxylates, polycations, masked oligo or poly cations or anions, acetals, polyacetals, ketals / polyketals, orthoesters, linear or branched polymers with masked orunmasked cationic or anionic charges, dendrimers with masked or unmasked cationic or anionic charges, polyanionic peptides, polyanionic peptidomimetics, pH-sensitive peptides, natural and synthetic fusogenic lipids, natural and synthetic cationic lipids.

[0172] Exemplary endosomolytic / fusogenic peptides include, but are not limited to, AALEALAEALEALAEALEALAEAAAAGGC (GALA, SEQ ID NO: 196);AALAEALAEALAEALAEALAEALAAAAGGC (EALA, SEQ ID NO: 197);ALEALAEALEALAEA (SEQ ID NO: 198); GLFEAIEGFIENGWEGMIWDYG (INF-7, SEQ ID NO: 199); GLFGAIAGFIENGWEGMIDGWYG (Inf HA-2, SEQ ID NO: 14);GLFEAIEGFIENGWEGMIDGWYGCGLFEAIEGFIENGWEGMID GWYGC (diINF-7, SEQ ID NO: 200); GLFEAIEGFIENGWEGMIDGGCGLFEAIEGFIENGWEGMIDGGC (diINF-3, SEQ ID NO: 201); GLFGALAEALAEALAEHLAEALAEALEALAAGGSC (GLF, SEQ ID NO: 202); GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC (GALA-INF3, SEQ ID NO: 203); GLF EAI EGFI ENGW EGnI DG K GLF EAI EGFI ENGW EGnI DG (INF-5, n is norleucine, SEQ ID NO: 204); LFEALLELLESLWELLLEA (JTS-1, SEQ ID NO: 205);GLFKALLKLLKSLWKLLLKA (ppTGl, SEQ ID NO: 206); GLFRALLRLLRSLWRLLLRA (ppTG20, SEQ ID NO: 207); WEAI<LAI<ALAI<ALAI<HLAI<ALAI<ALI<ACEA (KALA, SEQ ID NO: 208); GLFFEAIAEFIEGGWEGLIEGC (HA, SEQ ID NO: 209);GIGAVLKVLTTGLPALISWIKRKRQQ (Melittin, SEQ ID NO: 210); HsWYG (SEQ ID NO: 211); and CHKeHC (SEQ ID NO: 212).

[0173] Without wishing to be bound by theory, fusogenic lipids fuse with and consequently destabilize a membrane. Fusogenic lipids usually have small head groups and unsaturated acyl chains. Exemplary fusogenic lipids include, but are not limited to, l,2-dileoyl-sn-3- phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,31-tetraen- 19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-l,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 refered to as XTC herein).

[0174] Synthetic polymers with endosomolytic activity amenable to the present invention are described in U.S. Pat. App. Pub. Nos. 2009 / 0048410; 2009 / 0023890; 2008 / 0287630; 2008 / 0287628; 2008 / 0281044; 2008 / 0281041; 2008 / 0269450; 2007 / 0105804; 20070036865; and 2004 / 0198687, contents of which are hereby incorporated by reference in their entirety.

[0175] Exemplary cell permeation peptides include, but are not limited to, RQIKIWFQNRRMKWKK (penetratin, SEQ ID NO: 213); GRKKRRQRRRPPQC (Tat fragment 48-60, SEQ ID NO: 214); GALFLGWLGAAGSTMGAWSQPKKKRKV (signal sequence based peptide, SEQ ID NO: 215); LLIILRRRIRKQAHAHSK (PVEC, SEQ ID NO: 216);GWTLNSAGYLLKINLKALAALAKKIL (transportan, SEQ ID NO: 217);KLALKLALKALKAALKLA (amphiphilic model peptide, SEQ ID NO: 218); RRRRRRRRR (Arg9, SEQ ID NO: 219); KFFKFFKFFK (Bacterial cell wall permeating peptide, SEQ ID NO: 220); LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES (LL-37, SEQ ID NO: 221); SWLSKTAKKLENSAKKRISEGIAIAIQGGPR (cecropin Pl, SEQ ID NO: 222);ACYCRIPACIAGERRYGTCIYQGRLWAFCC (a-defensin, SEQ ID NO: 223);DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK (P-defensin, SEQ ID NO: 224); RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 (PR-39, SEQ ID NO: 225); ILPWKWPWWPWRR-NH2 (indohcidin, SEQ ID NO: 226); AAVALLPAVLLALLAP (RFGF, SEQ ID NO: 227); AALLPVLLAAP (RFGF analogue, SEQ ID NO: 228); and RKCRIVVIRVCR (bactenecin, SEQ ID NO: 229).

[0176] Exemplary cationic groups include, but are not limited to, protonated amino groups, derived from e.g., O- AMINE (AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH2)nAMINE, (e.g., AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); and NEI(CEECEENEI)nCEECEE-AMINE (AMINE = NEE; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino).

[0177] As used herein the term “targeting ligand” refers to any molecule that provides an enhanced affinity for a selected target, e.g., a cell, cell type, tissue, organ, region of the body, or a compartment, e.g., a cellular, tissue or organ compartment. Some exemplary targeting ligands include, but are not limited to, antibodies, antigens, folates, receptor ligands, carbohydrates, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDL ligands.

[0178] Carbohydrate based targeting ligands include, but are not limited to, D-galactose, multivalent galactose, N-acetyl-D-galactosamine (GalNAc), multivalent GalNAc, e.g. GalNAc2 and GalNAc3; D-mannose, multivalent mannose, multivalent lactose, N-acetyl-gulucosamine, multivalent fucose, glycosylated polyaminoacids and lectins. The term multivalent indicates that more than one monosaccharide unit is present. Such monosaccharide subunits can be linked to each other through glycosidic linkages or linked to a scaffold molecule.

[0179] A number of folate and folate analogs amenable to the present invention as ligands are described in U.S. Pat. Nos. 2,816,110; 5,552,545; 6,335,434 and 7,128,893, contents of which are herein incorporated in their entireties by reference.

[0180] As used herein, the terms “PK modulating ligand” and “PK modulator” refers to molecules which can modulate the pharmacokinetics of oligonucleotides described herein. Some exemplary PK modulator include, but are not limited to, lipophilic molecules, bile acids, sterols, phospholipid analogues, peptides, protein binding agents, vitamins, fatty acids, phenoxazine, aspirin, naproxen, ibuprofen, suprofen, ketoprofen, (S)-(+)-pranoprofen, carprofen, PEGs, biotin, and transthyretia-binding ligands (e.g., tetraiidothyroacetic acid, 2, 4, 6-triiodophenol and flufenamic acid). Oligomeric compounds that comprise a number of phosphorothioate intersugar linkages are also known to bind to serum protein, thus short oligomeric compounds, e.g. oligonucleotides of comprising from 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), and that comprise a plurality of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). The PK modulating oligonucleotide can comprise at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more phosphorothioate and / or phosphorodithioate linkages. In some embodiments, all intemucleoside linkages in PK modulating oligonucleotide are phosphorothioate and / or phosphorodithioates linkages. In addition, aptamers that bind serum components (e.g. serum proteins) are also amenable to the present invention as PK modulating ligands. Binding to serum components (e.g. serum proteins) can be predicted from albumin binding assays, scuh as those described in Oravcova, et al., Journal of Chromatography B (1996), 677: 1-27.

[0181] When two or more ligands are present, the ligands can all have same properties, all have different properties or some ligands have the same properties while others have different properties. For example, a ligand can have targeting properties, have endosomolytic activity or have PK modulating properties. In a preferred embodiment, all the ligands have different properties.

[0182] In some embodiments of any one of the aspects, the ligand has a structure shown in any of Formula (IV) - (VII):wherein: q2A, q2B, q3\ q3l\ q4 \ q4B, q5A, q5Band q5Crepresent independently for each occurrence0-20 and wherein the repeating unit can be the same or different; p2A p2B p3A p3B p4A p4B p5A p5B p5C y2A y2B y3A y3P> y4A y4B> y5A y5P> y5C each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH2, CH2NH or CH2O;Q2AQ2B Q3A Q3B Q4A Q4B Q5A QSB Q5Care mdependently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO2, N(RN), C(R’)=C(R”), C=C or C(O);R2AR2B,R3AR3BR4AR4B,R5ARSBR5Care eachindependently for each occurrence absent, NH, O, S, CH2, C(O)O, C(O)NH, NHCH(Ra)C(O), -C(O)-CH(Ra)-NH-, CO, CH=N-O,heterocyclyl;L2A, L2B, L3A, L3B, L4A, L4B, L5A, L5Band L5Crepresent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, tri saccharide, tetrasaccharide, oligosaccharide, or polysaccharide; andRais H or amino acid side chain.

[0183] In some embodiments of any one of the aspects, the ligand is of Formula (VII):wherein L5A, L5Band L5Crepresent a monosaccharide, such as GalNAc derivative.

[0184] Exemplary ligands include, but are not limited to, the following:Ligand 7Ligand 8.

[0185] In some embodiments of any one of the aspects described herein, the ligand is a ligand described in US Patent No. 5,994,517 or US Patent No. 6,906,182, content of each of which is incorporated herein by reference in its entirety.

[0186] In some embodiments, the ligand can be a tri-antennary ligand described in Figure 3 of US Patent No. 6,906,182. For example, the ligand is selected from the following tri-antennary ligands:

[0187] It is noted that when more than one ligand are present, they can be same or different. Accordingly, in some embodiments of any one of the aspects described herein, all ligands are same. In some other embodiments of any one of the aspects described herein, ligands are different.

[0188] In some embodiments of any one of the aspects described herein, the ligand is selected from the group consistof ligands shown in FIG. 27.Linkers

[0189] Embodiments of the various aspects described herein include a linker. As used herein, the term “linker” means an organic moiety that connects two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR1, C(O), C(O)O, C(O)NR1, SO, SO2, SO2NH or a chain of atoms, such as substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl. alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or more methylenes can be interrupted or terminated by O, S, S(O), SO2, N(R1)2, C(O), cleavable linking group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R1is hydrogen, acyl, aliphatic or substituted aliphatic.

[0190] In some embodiments, the linker is a cleavable linker. Cleavable linkers are those that rely on processes inside a target cell to liberate the two parts the linker is holding together, as reduction in the cytoplasm, exposure to acidic conditions in a lysosome or endosome, or cleavage by specific enzymes (e.g. proteases) within the cell. As such, cleavable linkers allow the two parts to be released in their original form after internalization and processing inside a target cell. Cleavable linkers include, but are not limited to, those whose bonds can be cleaved by enzymes (e.g., peptide linkers); reducing conditions (e.g., disulfide linkers); or acidic conditions (e.g., hydrazones and carbonates).

[0191] Generally, the cleavable linker comprises at least one cleavable linking group. A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times or more, preferably at least 100 times faster in the target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood or serum of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0192] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0193] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1- 7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0194] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, liver targeting ligands can be linked to the cationic lipids through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis. Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0195] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selectedto be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It may be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0196] One class of cleavable linking groups is redox cleavable linking groups, which may be used according to the present invention that are cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulfide linking group (-S-S-). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In a preferred embodiment, candidate compounds are cleaved by at most 10% in the blood. In preferred embodiments, useful candidate compounds are degraded at least 2, 4, 10 or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0197] Phosphate-based cleavable linking groups, which may be used in the dsRNA molecule according to the present invention, are cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, -O- P(S)(ORk)-O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O-P(O)(ORk)-S-, -S-P(O)(ORk)-S-, -O- P(S)(ORk)-S-, -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O-, -S-P(S)(Rk)- O-, -S-P(O)(Rk)-S-, -O-P(S)( Rk)-S-, wherein Rk at each occurrence can be, independently, hydrogen, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, C7-C12 aralkyl. Preferred embodiments are -O-P(O)(OH)-O-, -O-P(S)(OH)-O-, -O-P(S)(SH)-O-, -S-P(O)(OH)-O-, -O-P(O)(OH)-S-, -S- P(O)(OH)-S-, -O-P(S)(OH)-S-, -S-P(S)(OH)-O-, -O-P(O)(H)-O-, -O-P(S)(H)-O-, -S-P(O)(H)-O-,-S-P(S)(H)-O-, -S-P(O)(H)-S-, -O-P(S)(H)-S-. A preferred embodiment is -O-P(O)(OH)-O-. These candidates can be evaluated using methods analogous to those described above.

[0198] Acid cleavable linking groups, which may be used in the dsRNA molecule according to the present invention, are linking groups that are cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups 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). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0199] Ester-based cleavable linking groups, which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula - C(O)O-, or -OC(O)-. These candidates can be evaluated using methods analogous to those described above.

[0200] Peptide-based cleavable linking groups, which may be used in the dsRNA molecule according to the present invention, are cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynylene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula - NHCHRAC(O)NHCHRBC(O)-, where RAand RBare the R groups of the two adjacent amino acids.

[0201] In some embodiments of any one of the aspects described herein, the linker is - C(O)CH2CH2C(O)-, -OC(O)CH2CH2C(O)-, -OC(O)CH2CH2C(O)O-, -C(O)CH2CH2C(O)NH- or -OC(O)CH2CH2C(O)NH-. For example, the linker is -OC(O)CH2CH2C(O)NH-Internucleoside linkages

[0202] As used herein, “intemucleoside linkage” refers to a covalent linkage between adjacent nucleosides. The two main classes of intemucleoside linkages are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing linkages include, but are not limited to, phosphodiesters (P=O), phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing linking groups include, but are not limited to, methylenemethylimino ( — CH2-N(CH3)-O — CH2-), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ); siloxane ( — O — Si(H)2-0 — ); and N,N'- dimethylhydrazine ( — CH2-N(CH3)-N(CH3)-). Modified intemucleoside linkages, compared to natural phosphodiester linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide compound. In certain embodiments, linkages having a chiral atom can be prepared as racemic mixtures, as separate enantiomers. Representative chiral linkages include, but are not limited to, alkylphosphonates and phosphorothioates. Methods of preparation of phosphorous- containing and non-phosphorous-containing linkages are well known to those skilled in the art.

[0203] The phosphate group in the intemucleoside linkage can be modified by replacing one of the oxygens 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 phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphodiester intemucleoside linkage can be replaced by any of the following: S, Se, BR3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an aryl group, etc...), H, NR2 (R is hydrogen, optionally substituted alkyl, aryl), or OR (R is optionally substituted alkyl or aryl). The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms renders the phosphorous atom chiral. In other words a phosphorous atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp).

[0204] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligonucleotides diastereomers. Thus, while not wishing to be bound by theory, modifications to both non-bridging oxygens, which eliminate the chiral center, e.g. phosphorodithioate formation, can be desirable in that they cannot produce diastereomer mixtures. The non-bridging oxygens can be independently any one of O, S, Se, B, C, H, N, or OR (R is alkyl or aryl).

[0205] A phosphodiester intemucleoside linkage can also be modified by replacement of bridging oxygen, (i.e. oxygen that links the phosphate to the sugar of the nucleosides), with nitrogen(bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at the either one of the linking oxygens or at both linking oxygens. When the bridging oxygen is the 3 ’-oxygen of a nucleoside, replacement with carbon is preferred. When the bridging oxygen is the 5 ’-oxygen of a nucleoside, replacement with nitrogen is preferred.

[0206] Modified phosphate linkages where at least one of the oxygen linked to the phosphate has been replaced or the phosphate group has been replaced by a non-phosphorous group, are also referred to as “non-phosphodiester intersugar linkage” or “non-phosphodiester linker.”

[0207] In certain embodiments, the phosphate group can be replaced by non-phosphorus containing connectors, e.g. dephospho linkers. Dephospho linkers are also referred to as non- phosphodiester linkers herein. While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

[0208] Examples of moieties which can replace the phosphate group include, but are not limited to, amides (for example 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, carboxylate ester, thioether, ethylene oxide linker, sulfide, sulfonate, sulfonamide, sulfonate ester, thioformacetal (3'-S-CH2-O-5'), formacetal (3 '-O-CH2-O-5'), oxime, methyleneimino, methykenecarbonylamino, methyl enemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), methylenehydrazo, methylenedimethylhydrazo, methyleneoxymethylimino, ethers (C3’-O-C5’), thioethers (C3’-S-C5’), thioacetamido (C3’-N(H)-C(=O)-CH2-S-C5’, C3’-O-P(O)-O-SS-C5’, C3’- CH2-NH-NH-C5’, 3'-NHP(O)(OCH3)-O-5' and 3'-NHP(O)(OCH3)-O-5’ and nonionic linkages containing mixed N, O, S and CH2 component parts. See for example, Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65). Preferred embodiments include methylenemethylimino (MMI), methylenecarbonylamino, amides, carbamate and ethylene oxide linker.

[0209] One skilled in the art is well aware that in certain instances replacement of a non- bridging oxygen can lead to enhanced cleavage of the intersugar linkage by the neighboring 2’- OH, thus in many instances, a modification of a non-bridging oxygen can necessitate modification of 2’-OH, e.g., a modification that does not participate in cleavage of the neighboring intersugar linkage, e.g., arabinose sugar, 2’-O-alkyl, 2’-F, LNA and ENA.

[0210] Preferred non-phosphodiester intemucleoside linkages include phosphorothioates, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%95% or more enantiomeric excess of Sp isomer, phosphorothioates with an at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90% 95% or more enantiomeric excess of Rp isomer, phosphorodi thioates, phsophotriesters, aminoalky Iphosphotrioesters, alkyl-phosphonaters (e.g., methyl-phosphonate), selenophosphates, phosphorami dates (e.g., N-alkylphosphoramidate), and boranophosphonates.

[0211] Additional exemplary non-phosphorus containing intemucleoside linking groups are described in U.S. Patent 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; 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, content of each of which is incorporated herein by reference.

[0212] In some embodiments of any one of the aspects, the oligonucleotides described herein comprise one or more neutral intemucleoside linkages that are non-ionic. Suitable neutral intemucleoside linkages include, but are not limited to, phosphotriesters, methylphosphonates, 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'); nonionic linkages containing siloxane (dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonate ester and / or amides (See for example: Carbohydrate Modifications in Antisense Research; Y.S. Sanghvi and P.D. Cook Eds. ACS Symposium Series 580; Chapters 3 and 4, (pp. 40-65)); and nonionic linkages containing mixed N, O, S and CEE component parts.

[0213] In one embodiment, the non-phosphodiester backbone linkage is selected from the group consisting of phosphorothioate, phosphorodithioate, alkyl-phosphonate and phosphoramidate backbone linkages.

[0214] In some embodiments of any one of the aspects described herein, the intemucleoside linkagewhere RIL1and RIL2are each independently for each occurrence absent, O, S, CEE, NR (R is hydrogen, alkyl, aryl), or optionally substituted alkylene, wherein backbone of the alkylene can comprise one or more of O, S, SS and NR (R is hydrogen, alkyl, aryl) internally and / or at the end; and RIL3and RIL4are each independently selected from the group consisting of O, OR (R is hydrogen, alkyl, aryl), S, Se, BR3 (R is hydrogen, alkyl, aryl), BEE' , C (i.e. an alkyl group, an aryl group, etc... ), H, NR2(R is hydrogen, alkyl, aryl), alkyl or aryl. It is understood that one of RIL1and RIL2is replacing the oxygen linked to 5’ carbon of a firstnucleoside sugar and the other of RIL1and RIL2is replacing the oxygen linked to 3’ (or 2’) carbon of a second nucleoside sugar.

[0215] In some embodiments of any one of the aspects, RIL1, RIL2, RIL3and RIL4all are O.

[0216] In some embodiments, RIL1and RIL2are O and at least one of RIL3and RIL4is other thanO. For example, one of RIL3and RIL4is S and the other is O or both of RIL3and RIL4are S.

[0217] In some embodiments of any one of the aspects described herein, one of R43or R45is a bond to a modified intemucleoside linkage, e.g., an intemucleoside linkage of structure:where at least one of RIL1, RIL2, RIL3and RIL4is not O. For example, at least one of RIL3and RIL4is S.

[0218] In some embodiments of any one of the aspects described herein, both of R43and R45are a bond to a modified intemucleoside linkage.

[0219] In some embodiments of any one of the aspects described herein R43is a bond to phosphodiester intemucleoside linkage.

[0220] In some embodiments of any one of the aspects described herein R45is a bond to phosphodiester intemucleoside linkage.

[0221] In some embodiments of any one of the aspects described herein, R43is a bond to a modified intemucleoside linkage and R45is a bond to phosphodiester intemucleoside linkage.

[0222] In some embodiments of any one of the aspects described herein, R45is a bond to a modified intemucleoside linkage and R43is a bond to phosphodiester intemucleoside linkage.

[0223] In some embodiments of any one of the aspects described herein, the intemucleotide linkage is -P(XD)(N(Rp)2)-, where XDis O or S; and each RP2is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl.

[0224] In some embodiments of any one of the aspects described herein, R43is linked to an intemucleotide linkage of formula -P(XD)(N(Rp)2)-@, where XDis O or S; each RP2is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to 5 ’-position of a subsequent nucleoside. For example, R43is linked to an intemucleotide linkage of formula -P(XD)(N(Rp)2)-@, where XDis O or S; each RP2is independently an optionally substituted alkyl, e.g., Ci-ealkyl, such as methyl; and @ is a bond to R45of a subsequent nucleoside of Formula (IV).

[0225] In some embodiments of any one of the aspects, the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more modified intemucleoside linkages. For example, the oligonucleotide can comprise 1, 2, 3, 4, 5 or 6 modified intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3 or 4 modified intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two modified intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide. For example, the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.

[0226] In some embodiments of any one of the aspects, the modified intemucleoside linkage is a phosphorothioate. Accordingly, in some embodiments of any one of the aspects, the oligonucleotide comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioate intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3, 4, 5 or 6 phosphorothioate intemucleoside linkages. For example, the oligonucleotide comprises 1, 2, 3 or 4 phosphorothioate intemucleoside linkages. In some embodiments, the oligonucleotide comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the oligonucleotide and further comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the oligonucleotide. For example, the oligonucleotide comprises modified intemucleoside linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the oligonucleotide, and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the oligonucleotide.Oxygen protecting groups

[0227] Some embodiments of the various aspects described herein include an oxygen protecting group (also referred to as an hydroxyl protecting group herein). Oxygen protecting groups include, but are not limited to, -ROP1, -N(ROP2)2, -C(=O)SRopl, -C(=O)Ropl, -CO2Ropl, -C(=O)N(ROP2)2, -C(=NROP2)ROP1, -C(=NR°P2)OROP1, -C(=NROP2)N(ROP2)2, -S(=O)ROP1, -SO+2ROP1, -SI(ROP1)3, -P(ROP3)2, -P(ROP3)+3 X , -P(OROP3)2, -P(OROP3)3 X , -P(=O)(ROP1)2, -P(=O)(OR°P3)2, and -P(=O)(N(ROP2)2)2; wherein each X is a counterion; each ROP1is independently Ci-io alkyl, Ci-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi-io alkyl, heteroC2-ioalkenyl, heteroC2-ioalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, or 5-14 membered heteroaryl, or two ROP1groups are joined to form a 3-14 membered heterocyclylor 5-14 membered heteroaryl ring; each ROP2is hydrogen, -OH, -ORopl, -N(ROP3)2, -CN, -C(=O)Ropl, -C(=O)N(ROP3)2, -CO2ROP1, -SO2ROP1, -C(=NR°P3)OROP1, -C(=NROP3)N(ROP3)2, -SO2N(ROP3)2, -SO2ROP3, -SO2OR°P3, -SOROP1, -C(=S)N(ROP3)2, -C(=O)SROP3, -C(=S)SROP3, -P(=O)(ROP1)2, -P(=O)(OROP3)2, -P(=O)(N(ROP3)2)2, CI-10 alkyl, Ci-io perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi- walkyl, heteroC2-ioalkenyl, heteroC2-ioalkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two ROP2groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each ROP3is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroCi- 10 alkyl, heteroC2-io alkenyl, heteroC2-io alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, Ce-14 aryl, and 5-14 membered heteroaryl, or two ROP3groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of ROP1, ROP2and ROP3can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=0), SH, SO2NH2, SO2(Ci-C4)alkyl, SO2NH(Ci-C4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(Ci-C4)alkyl, N[(Ci-C4)alkyl]2, C(0)NH2, COOH, COOMe, acetyl, (Ci-Cs)alkyl, O(Ci- Csjalkyl (i.e., Ci-Csalkoxy), O(Ci-Cs)haloalkyl, (C2-Cs)alkenyl, (C2-Cs)alkynyl, haloalkyl, thioalkyl, cyanomethylene, alkylaminyl, aryl, heteroaryl, substituted aryl, NH2 — C(O)-alkylene, NH(Me)-C(O)-alkylene, CH2 — C(0)- alkyl, C(0)- alkyl, alkylcarbonylaminyl, CH2 — [CH(0H)]m— (CH2)P— OH, CH2— [CH(0H)]m— (CH2)P— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0228] Oxygen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5thEdition, John Wiley & Sons, 2014, incorporated herein by reference.

[0229] Exemplary oxygen protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boc), methoxylmethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (TEIP), 3 -bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP), 4-methoxytetrahydrothiopyranyl, 4- methoxytetrahydrothiopyranyl S,S-dioxide, 1 -[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4- yl (CTMP), l,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro- 7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1 -ethoxy ethyl, 1 -(2-chloroethoxy)ethyl, 1-methyl-l- methoxy ethyl, 1 -methyl- 1 -benzyloxy ethyl, 1- methyl- 1 -benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o- nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2- picolyl, 4-picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p'-dinitrobenzhydryl, 5- dibenzosuberyl, triphenylmethyl, a-naphthyldiphenylmethyl, p-methoxyphenyldiphenylmethyl, di(p- methoxyphenyl)phenylmethyl, tri(p-methoxyphenyl)methyl, 4-(4'- bromophenacyloxyphenyl)diphenylmethyl, 4,4',4"-tris(4,5-dichlorophthalimidophenyl)methyl, 4,4',4"-tris(levulinoyloxyphenyl)methyl, 4, 4', 4"- tris(benzoyloxyphenyl)methyl, 3-(imidazol-l- yl)bis(4',4"-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-l'-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, l,3-benzodisulfuran-2-yl, benzisothiazolyl S,S- dioxido, trimethylsilyl (TMS), triethylsilyl (TES), triisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate, acetate, chloroacetate, dichloroacetate, trichloroacetate, trifluoroacetate, methoxyacetate, triphenylmethoxyacetate, phenoxyacetate, p- chlorophenoxyacetate, 3 -phenylpropionate, 4- oxopentanoate (levulinate), 4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate, 2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate, 9- fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec), 2- (triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkyl p-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzyl carbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzyl carbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate, 4-ethoxy-l-napththyl carbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate, 2- formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl, 4- (methylthiomethoxy)butyrate, 2- (methylthiomethoxymethyl)benzoate, 2,6-dichloro-4- methylphenoxyacetate, 2,6-dichloro-4- (1,1 ,3,3-tetramethylbutyl)phenoxyacetate, 2,4- bis( 1 , 1 -dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate, monosuSP3inoate, (E)-2-methyl-2-butenoate, o- (methoxyacyl)benzoate, a-naphthoate, nitrate, alkylN,N,N',N'-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0230] In some embodiments of any one of the aspects described herein, oxygen protecting group is benzyl, benzoyl, 2,6-dichlorobenzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, mesylate, tosylate, 4,4'-dimethoxytrityl (DMT), 9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl (MOX). In certain embodiments, the hydroxyl protecting group is selected from acetyl, benzyl, t-butyldimethylsilyl, t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4′-dimethoxytrityl.

[0231] The terms “protected hydroxyl” and “protected hydroxyl” as used herein mean a group of the formula -ORPro, wherein RProis an oxygen protecting group as defined herein. Nitrogen protecting groups

[0232] Some embodiments of the various aspects described herein include a nitrogen protecting group (also referred to as an amino protecting group herein). Nitrogen protecting groups include, but are not limited to, -OH, -ORNP1, -N(RNP2)2, -C(=O)RNP1, -C(=O)N(RNP2)2, -CO2RNP1, - SO2RNP1, -C(=NRNP2)RNP1, -C(=NRNP2)ORNP1, -C(=NRNP2)N(RNP2)2, -SO2N(RNP2)2, -SO2RNP2, - SO2ORNP2, -SORNP1, -C(=S)N(RNP2)2, -C(=O)SRNP2, -C(=S)SRNP2, C1-10 alkyl (e.g., aralkyl, heteroaralkyl), C2-10alkenyl, C2-10alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl groups, where each RNP1is independently C1-10alkyl, C1-10perhaloalkyl, C2-10alkenyl, C2-10alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RNP1groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RNP2is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14aryl, and 5-14 membered heteroaryl, or two RSP3groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RNP1and RNP2can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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—NH2 or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0233] Nitrogen protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5thEdition, John Wiley & Sons, 2014, incorporated herein by reference.

[0234] Exemplary amide (e.g., -C(=O)RNP1) nitrogen protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide, 3 -pyridylcarboxamide, N- benzoylphenylalanyl derivative, benzamide, p- phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N'- dithiobenzyloxy acylamino)acetamide, 3-(p- hy droxylphenyl)propanamide, 3 -(o-nitrophenyl)propanamide, 2-methyl-2-(o- nitrophenoxy)propanamide, 2-methyl-2-(o- phenylazophenoxy)propanamide, 4- chlorobutanamide, 3-methyl-3-nitrobutanamide, o- nitrocinnamide, N-acetylmethionine derivative, o-nitrobenzamide, and o-(benzoyloxymethyl)benzamide.

[0235] Exemplary carbamate (e.g., -C(=O)ORNP1) nitrogen protecting groups include, but are not limited to, methyl carbamate, ethyl carbamate, 9-fluorenylmethyl carbamate (Fmoc), 9-(2- sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate, 2,7-di-t-butyl-[9- (10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methyl carbamate (DBD-Tmoc), 4- methoxyphenacyl carbamate (Phenoc), 2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate (Teoc), 2-phenylethyl carbamate (hZ), 1- (l-adamantyl)-l-methylethyl carbamate (Adpoc), l,l-dimethyl-2-haloethyl carbamate, 1 , 1 -dimethyl-2,2-dibromoethyl carbamate (DB-t- BOC), l,l-dimethyl-2, 2, 2-tri chloroethyl carbamate (TCBOC), 1 -methyl- l-(4-biphenylyl)ethyl carbamate (Bpoc), l-(3,5-di-t- butylphenyl)-! -methylethyl carbamate (t-Bumeoc), 2-(2'- and 4'- pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethyl carbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc), vinyl carbamate (Voc), allyl carbamate (Alloc), 1- isopropylallyl carbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc), 8-quinolyl carbamate, N-hydroxylpiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz), p-nitobenzyl carbamate, p- bromobenzyl carbamate, p-chlorobenzyl carbamate, 2,4-dichlorobenzyl carbamate, 4- methylsulfinylbenzyl carbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate, 2- methylthioethyl carbamate, 2-methylsulfonylethyl carbamate, 2-(p- toluenesulfonyl)ethyl carbamate, [2-(l,3-dithianyl)]methyl carbamate (Dmoc), 4- methylthiophenyl carbamate (Mtpc), 2,4-dimethylthiophenyl carbamate (Bmpc), 2- phosphonioethyl carbamate (Peoc), 2- triphenylphosphonioisopropyl carbamate (Ppoc), 1,1- dimethyl-2-cyanoethyl carbamate, m- chloro-p-acyloxybenzyl carbamate, p-(dihydroxylboryl)benzyl carbamate, 5- benzisoxazolylmethyl carbamate, 2-(trifluoromethyl)- 6-chromonylmethyl carbamate (Tcroc), m- nitrophenyl carbamate, 3, 5 -dimethoxybenzyl carbamate, o-nitrobenzyl carbamate, 3,4-dimethoxy- 6-nitrobenzyl carbamate, phenyl(o- nitrophenyl)methyl carbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzyl carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate, 2,2- dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzyl carbamate, l,l-dimethyl-3- (N,N- dimethylcarboxamido)propyl carbamate, 1 , 1 -dimethylpropynyl carbamate, di (2-pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p'-methoxyphenylazo)benzyl carbamate, 1 -methylcyclobutyl carbamate, 1 -methylcyclohexyl carbamate, 1 -methyl- 1- cyclopropylmethyl carbamate, l-methyl-l-(3,5-dimethoxyphenyl)ethyl carbamate, 1- methyl-l-(p- phenylazophenyl)ethyl carbamate, 1-methyl-l -phenylethyl carbamate, 1- methyl-l-(4- pyridyl)ethyl carbamate, phenyl carbamate, p-(phenylazo)benzyl carbamate, 2,4,6-tri-t- butylphenyl carbamate, 4-(trimethylammonium)benzyl carbamate, and 2,4,6- trimethylbenzyl carbamate.

[0236] Exemplary sulfonamide (e.g., -S(=O)2RNP1) nitrogen protecting groups include, but are not limited to, such as p-toluenesulfonamide (Ts), benzenesulfonamide, 2,3,6, - trimethyl-4- methoxybenzenesulfonamide (Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb), 2,6-dimethyl-4- methoxybenzenesulfonamide (Pme), 2,3,5,6-tetramethyl-4- methoxybenzenesulfonamide (Mte), 4- methoxybenzenesulfonamide (Mbs), 2,4,6- trimethylbenzenesulfonamide (Mts), 2,6-dimethoxy-4- methylbenzenesulfonamide (iMds), 2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide (Ms), 0- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4',8'-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0237] Additional exemplary nitrogen protecting groups include, but are not limited to, phenothiazinyl-(10)-acyl derivative, N'-p-toluenesulfonylaminoacyl derivative, N'- phenylaminothioacyl derivative, N-benzoylphenylalanyl derivative, N-acetylmethionine derivative, 4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuNP2inimide (Dts), N- 2,3- diphenylmaleimide, N-2,5-dimethylpyrrole, N-l,l,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted l,3-dimethyl-l,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-l,3,5-triazacyclohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N- allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3 -acetoxypropylamine, N-(l- isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N- di(4- methoxyphenyl)methyl amine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fem), N-2- picolylamino N'- oxide, N- 1,1 -dimethylthiomethyleneamine, N-benzylideneamine, N-p- methoxybenzylideneamine, N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl] methyleneamine, N-(N',N'-dimethylaminomethylene)amine, N,N'- isopropylidenediamine, N-p- nitrobenzylideneamine, N-salicylideneamine, N-5- chlorosalicylideneamine, N-(5-chloro-2- hydroxylphenyl)phenylmethyleneamine, N- cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-l- cyclohexenyl)amine, N-borane and N-diphenylborinic acid derivative, N-[phenyl(pentNP1cylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N- nitroamine, N-nitrosoamine, amine N-oxide, diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt), diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl phosphoramidate, benzenesulfenamide, o- nitrobenzenesulfenamide (Nps), 2,4- dinitrobenzenesulfenamide, pentachlorobenzenesulfenamide, 2-nitro-4- methoxybenzenesulfenamide, triphenylmethylsulfenamide, and 3- nitropyridinesulfenamide (Npys). Sulfur protecting groups

[0238] Some embodiments of the various aspects described herein include sulfur protecting group (also referred to as a thiol protecting group herein). Sulfur protecting groups include, but are not limited to, -RSP1, -N(RSP2)2, -C(=O)SRSP1, -C(=O)RSP1, -CO2RSP1, −C(=O)N(RSP2)2, - C(=NRSP2)RSP1, -C(=NRSP2)ORSP1, -C(=NRSP2)N(RSP2)2, -S(=O)RSP1, -SO2RSP1, −Si(RSP1)3, - P(RSP3)2, -P(RSP3)+3 X−, -P(ORSP3)2, -P(ORSP3)+3 X−, -P(=O)(RSP1)2, -P(=O)(ORSP3)2, and−P(=O)(N(RSP2) 2)2, wherein

[0239] X- is a counterion; each RSP1is independently C1-10 alkyl, C1-10 perhaloalkyl, C2- 10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3- 10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, or 5-14 membered heteroaryl, or two RSP1groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; each RSP2is hydrogen, −OH, −ORSP1, −N(RSP3)2, −CN, −C(=O)RSP1, −C(=O)N(RSP3)2, −CO2RSP1, −SO2RSP1, −C(=NRSP3)ORSP1, −C(=NRSP3)N(RSP3)2, −SO2N(RSP3)2, −SO2RSP3, −SO2ORSP3, −SORSP1, −C(=S)N(RSP3)2, −C(=O)SRSP3, −C(=S)SRSP3, −P(=O)(RSP1)2, −P(=O)(ORSP3)2, −P(=O)(N(RSP3)2)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP2groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RSP3is independently hydrogen, C1-10alkyl, C1-10perhaloalkyl, C2-10alkenyl, C2-10alkynyl, heteroC1-10alkyl, heteroC2-10alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two RSP3groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl, and heteroaryl of RSP1, RSP2and RSP3can be optionally substituted with 1, 2, 3, 4 or 5 substituents independently selected from 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-C8alkoxy), 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— NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6.

[0240] Sulfur protecting groups are well known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5thEdition, John Wiley & Sons, 2014, incorporated herein by reference.

[0241] It is noted that the nucleoside of Formula (IV) can be located anywhere in the oligonucleotide. In some embodiments, the nucleoside of Formula (IV) is present at the 5’- or 3’- terminus of the oligonucleotide. In some embodiments, the nucleoside of Formula (IV) is present at an internal position of the oliogunculeotide. In some embodiments, when the nucleoside of Formula (IV) is present at the 3 ’-terminus of the oligonucleotide, R43is a hydroxyl or protected hydroxyl group. In some embodiments, when the nucleoside of Formula (IV) is present at the 3’- terminus of the oligonucleotide, R43is a hydroxyl. In other embodiments, when the nucleoside of Formula (IV) is present at the 3 ’-terminus of the oligonucleotide, R43is a hydrogen or a nitrogen protecting group. In other embodiments, when the nucleoside of Formula (IV) is present at the 3’- terminus of the oligonucleotide, R43is a hydrogen.

[0242] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a nucleoside with a modified sugar. By a “modified sugar” is meant a sugar or moiety other than 2’-deoxy (i.e, 2’-H) or 2’-OH ribose sugar. Some exemplary nucleotides comprising a modified sugar are 2’-F ribose, 2’-0Me 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'-O-N-methylacetamido (2'-0-NMA) ribose, a 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 is noted that the nucleoside with the modified sugar can be present at any position of the oligonucleotide.

[0243] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 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. It is noted that the 2’-F nucleotides can be present at any position of the oligonucleotide.

[0244] In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), and 2’-F nucleosides.

[0245] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-0Me nucleotides. For example, the oligonucleotide can comprise 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-0Me nucleotides. It is noted that the 2’-0Me nucleotides can be present at any position of the oligonucleotide.

[0246] In some embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises solely comprises nucleosides of Formula (IV), and 2’-0Me nucleosides. In some other embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides and 2’-F nucleosides.

[0247] In some embodiments, the oligonucleotide further comprises at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 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 of 2’-deoxy, e.g., 2’-H nucleotides. It is noted that the 2’- deoxy, e.g., 2’-H nucleotides can be present at any position of the oligonucleotide. For example, the oligonucleotide can comprise a 2’-deoxy, e.g., 2’-H nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the oligonucleotide. In some embodiments, the oligonucleotide comprises a 2’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the oligonucleotide.

[0248] In some embodiments, the oligonucleotide comprises, e.g., solely comprises solely comprises nucleosides of Formula (IV), and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides, and 2’-deoxy (2’-H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-F nucleosides and 2’-deoxy (2’- H) nucleotides. In some embodiments, the oligonucleotide comprises, e.g., solely comprises nucleosides of Formula (IV), 2’-OMe nucleosides, 2’-F nucleosides and 2’-deoxy (2’-H) nucleotides.

[0249] In some embodiments of any one of the aspects described herein, the oligonucleotide further comprises, i.e., in addition to a nucleotiside of Formula (IV), a non-natural nucleobase. In some embodiments, the oligonucleotide can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising an independently selected non-natural nucleobase. When present, a nucleotide comprising a non-natural nucleobase can be present anywhere in the oligonucleotide.

[0250] In some embodiments, the oligonucleotide further comprises a solid support linked thereto.

[0251] The oligonucleotides described herein can range from few nucleotides (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides) in length to hunderes of nucleotides in length. For example, the oligonucleotide can be from 5 nucleotides to 100 nucleotides in length. In some embodiments, the oligonucleotide is from 10 nucleotides to 50 nucleotides in length. For example, the oligonucleotide is between 15 and 35, more generally between 18 and 25, yet more generally between 19 and 24, and most generally between 19 and 21 nucleotides in length. In someembodiments, longer oligonucleotides of between 25 and 30 nucleotides in length are preferred. In some embodiments, shorter oligonucleotides of between 10 and 15 nucleotides in length are preferred. In another embodiment, the oligonucleotide is at least 21 nucleotides in length.

[0252] In some embodiments, the oligonucleotide described herein comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

[0253] In some embodiments, the oligonucleotide described herein comprises a stereochemistry block. In some embodiments, a block is an Rp block in that each intemucleotidiclinkage of the block is Rp. In some embodiments, a 5 ’-block is an Rp block. In some embodiments, a 3 ’-block is an Rp block. In some embodiments, a block is an Sp block in that each intemucleotidic linkage of the block is Sp. In some embodiments, a 5 ’-block is an Sp block. In some embodiments, a 3 ’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each intemucleotidic linkage in a natural phosphate linkage.

[0254] In some embodiments, the oligonculeotide described herein comprises a 5’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification. In some embodiments, a 5 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 5’- block is an Sp block wherein each of intemucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 5’-block comprises 4 or more nucleoside units. In some embodiments, a 5 ’-block comprises 5 or more nucleoside units. In some embodiments, a 5 ’-block comprises 6 or more nucleoside units. In some embodiments, a 5 ’-block comprises 7 or more nucleoside units. In some embodiments, a 3 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block comprises 4 or more nucleoside units. In some embodiments, a 3 ’-block comprises 5 or more nucleoside units. In some embodiments, a 3 ’-block comprises 6 or more nucleoside units. In some embodiments, a 3 ’-block comprises 7 or more nucleoside units.

[0255] In some embodiments, oligonucleotide described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of intemucleotidic linkage, e.g., natural phosphate linkage, modified intemucleotidic linkage, Rp chiral intemucleotidic linkage, Sp chiral intemucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G isfollowed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0256] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'-monophosphate ((HO)2(O)P-O-5'); 5 '-diphosphate ((HO)2(O)P- O-P(HO)(O)-O-5'); 5'-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'- adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-51- (HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-monothiophosphate (phosphorothioate; (HO)2(S)P- 0-5'); 5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5 '-alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5'- phosphoramidates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (e.g., RP(OH)(O)-O-5'-, R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e. vinyl, substituted vinyl, e.g., OH)2(O)P-5'-CH= or (OH)2(O)P-5'-CH2-), 5'- alkyletherphosphonates (e.g., R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.) Other exemplary 5 ’-modifications include where Z is optionally substituted alkyl at least once, e g., ((HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH2)a- P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: HO[-(CH2)a-O-P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[- (CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(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 a and b are each independently 1-10. Other embodiments, include replacement of oxygen and / or sulfur with BH3, BH?" and / or Se.

[0257] In some embodiments of any one of the aspects described herein, the oligonucleotide comprises a 5’-vinylphosphonate group (i.e., the 4’-C of the 5’-terminal nucleotide is bonded to a vinyl phosphonate). For example, the oligonucleotide comprises a 5’-E-vinyl phosphonate group. In some other non-limiting example, the oligonucleotide comprises a 5’-Z-vinylphosphonate group.

[0258] In some embodiments of any one of the aspects, the oligonucleotide dscribed herein comprises a 5 ’-morpholino, a 5 ’-dimethylamino, a 5 ’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5 ’-end.

[0259] In some embodiments of any one of the aspects, the oligonucleotide dscribed herein can comprise a thermally destabilizing modification, for example, a nucleoside of formula (IV), within the seed region of the antisense strand. For example, the oligonucleotide can comprise at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5’-end of the oligonucleotide (e.g., one thermally destabilizing nucleotide). In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand. In some embodiments, thermally destabilizing modification is located in positions 2-9, or preferably positions 4-8, counting from the 5 ’-end of the oligonucleotide. In some further embodiments, the thermally destabilizing modification is located at position 5, 6, 7 or 8, counting from the 5’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 7, counting from the 5 ’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 6, counting from the 5 ’-end of the oligonucleotide. In still some further embodiments, the thermally destabilizing modification is located at position 5, counting from the 5 ’-end of the oligonucleotide.

[0260] The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, counting from the 5 ’-end of the antisense strand.

[0261] The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA). For example, the thermally destabilizing modifications can include, but are not limited to, mUNA and GNA building blocks as follows:

[0262] 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.

[0263] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of- alkyl; O-alkylamino;R' = H, Me;B = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; andStereochemistry is R or S and combination of R and S for the unspecified chiral centers.

[0264] In some embodiments, the destabilizing modification mUNA is selected from the group consisting of; O- alkyl; O-alkylamino;R' = H, Me;B = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines;C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi andtricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; and Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.

[0265] In some embodiments, the destabilizing modification mUNA is selected from the group consisting ofB = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurines; and Stereochemistry is R or S and combination of R and S for the unspecified chiral centers.

[0266] In some embodiments, the destabilizing modification mUNA is selected from the group consisting ofR = H, OH; OMe; Cl, F; OH; O-(CH2)2OMe; SMe, NMe2; NH2; Me; CCH (alkyne), O-wPr; O- alkyl; O-alkylamino;R' = H, Me;B = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; andStereochemistry is R or S and combination of R and S for the unspecified chiral centers

[0267] In some embodiments, the destabilizing modification mUNA is selected from the group consisting ofalkyl; O-alkylamino;R' = H, Me;B = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; 7-deazapurines, phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; andStereochemistry is R or S and combination of R and S for the unspecified chiral centers

[0268] In some embodiments, the modification mUNA is selected from the group consisting ofB = A; C; 5-Me-C; G; I; U, 5-MeU; T; Y; 2-thiouridine; 4-thiouridine; C5-modified pyrimidines; C2-modified purines; N8-modiifed purines; phenoxazine; G-clamp; non-canonical mono, bi and tricyclic heterocycles; pseudouracil; isoC; isoG; 2,6-diamninopurine; pseudocytosine; 2- aminopurine; xanthosine; N6-alkyl-A; O6-alkyl-G; 7-deazapurines; andStereochemistry is R or S and combination of R and S for the unspecified chiral centers

[0269] Exemplary abasic modifications include, but are not limited to the following:Mod3 Mod4 Mod5 (2'-OMe Abasic (3'-0Me) (5'-Me) (Hyp-spacer) Spacer)X = OMe, F wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

[0270] Exemplified sugar modifications include, but are not limited to the following:R= H, OH, O-alkyl R'" = H, OH, CH3, CH2CH3, O-alkyl, NH2, NHMe, NMe2R"" = H, OH, CH3, CH2CH3, O-alkyl, NH2, NHMe, NMe2wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

[0271] In some embodiments the thermally destabilizing modification of the duplex is selected from the mUNA and GNA building blocks described in Examples 1-3 herein. 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. In some further embodiments of this, the dsRNA molecule further comprises at least one thermally destabilizing modification selected from the group consisting of GN A, 2’-0Me, 3’-0Me, 5 ’-Me, Hy p-spacer, SNA, hGNA, hhGNA, mGNA, TNA and h’GNA (Mod A-Mod K).

[0272] The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., Cl’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or Cl’-O4’) is absent and / or at least one of ribose carbons or oxygen (e.g., Cl’, C2’, C3’,C4’ or 04’) are independently or in combination absent from the nucleotide. In some, independently are H, halogen, OR3, or alkyl; andR3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between CT-C4' being removed (i.e. the covalent carbon- oxygen-carbon bond between the Cl' and C4' carbons). In another example, the C2'-C3' bond (i.e. the covalent carbon-carbon bond between the C2' and C3' carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage.

[0273] The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:(R)-GNA

[0274] The thermally destabilizing modification of the duplex can be mismatches (i.e., noncompl ementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, GA, GU, G:T, A: A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2’-deoxy nucleobase; e.g., the 2’-deoxy nucleobase is in the sense strand.

[0275] In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:

[0276] More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011 / 133876, which is herein incorporated by reference in its entirety.

[0277] The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

[0278] In some embodiments, the thermally destabilizing modification includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010 / 0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:inosine nebularine 2-aminopurine2,4- difluorotoluene 5-nitroindole 3-nitropyrrole 4-Fluoro-6- 4-Methylbenzimidazole methylbenzimidazole

[0279] In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more a-nucleotide complementary to the base on the target mRNA, such as:wherein R is H, OH, OCH3, F, NH2, NHMe, NM02 or O-alkyl

[0280] Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:

[0281] The alkyl for the R group can be a Ci-Cealkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

[0282] In some embodiments of any one of the aspects described herein, the oligonucleotide can comprise one or more stabilizing modifications. For example, the oligonucleotide can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications.

[0283] In some embodiments, the oligonucleotide comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the oligonucleotide can be present at any positions. In some embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16, counting from the 5 ’-end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 6, 14 and 16, counting from the 5 ’-end. In still some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 2, 14 and 16, counting from the 5 ’-end. In some embodiments, the oligonucleotide comprises stabilizing modifications at positions 7, 10 and 11, counting from the 5 ’-end. In some other embodiments, the oligonucleotide comprises stabilizing modifications at positions 7, 9, 10 and 11, counting from the 5 ’-end.

[0284] In some embodiments, the oligonucleotide comprises at least one stabilizing modification adjacent to a destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5 ’-end or the 3 ’-end of the destabilizing modification, i.e., at position -1 or+1 from the position of the destabilizing modification. In some embodiments, the oligonucleotide comprises a stabilizing modification at each of the 5 ’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.

[0285] In some embodiments, the oligonucleotide comprises at least two stabilizing modifications at the 3 ’-end of a destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0286] Exemplary thermally stabilizing modifications include, but are not limited to 2’-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to LNA.Double-stranded RNAs

[0287] The skilled person is well aware that double-stranded RNAs comprising a duplex structure of between 20 and 23, but specifically 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer double-stranded oligonucleotides can be effective as well.

[0288] Accordingly, in one aspect, provided herein is a double-stranded RNA (dsRNA) comprising a first strand (also referred to as an antisense strand or a guide strand) and a second strand (also referred to as a sense strand or passenger strand, wherein at least one of the first (i.e., the antisense strand) or the second strand (i.e., the sense strand) is an oligonucleotide described herein. In other words, at least one of the first (i.e., the antisense strand) or the second strand (i.e., the sense strand) comprises at least one nucleotide of Formula (IV),

[0289] 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 (IV),

[0290] 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 (IV).

[0291] 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 mRNA gene and the dsRNA is capable of inducing targeted cleavage of the target nucleic acid.

[0292] Each strand of the dsRNA molecule can range from 15-35 nucleotides in length. For example, each strand can be between, 17-35 nucleotides in length, 17-30 nucleotides in length, 17- 25 nucleotides in length, 18-30 nucleotides in length 18-25 nucleotides in length, 25-35 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17- 19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. Without limitations, the senseand antisense strands can be equal length or unequal length. For example, the sense strand and the antisense strand independently have a length of 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides.

[0293] In some embodiments, the antisense strand is of length 15-35 nucleotides. In some embodiments, the antisense strand is 15-35, 17-35, 17-30, 17-25, 18-30, 18-25, 25-35, 27-30, 17- 23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the antisense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 nucleotides in length. In some embodiments, the antisense strand is 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the antisense strand is 22, 23 or 24 nucleotides in length. For example, the antisense strand is 23 nucleotides in length.

[0294] Similar to the antisense strand, the sense strand can be, in some embodiments, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 17-25 nucleotides in length, 18-30 nucleotides in length 18-25 nucleotides in length, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense strand can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or35 nucleotides in length. In some embodiments, the sense strand is 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides in length. For example, the sense strand is 21nucleotides in length.

[0295] In some embodiments, the sense strand can be 15-35 nucleotides in length, and the antisense strand can be independent from the sense strand, 15-35 nucleotides in length. In some embodiments, the sense strand is 15-35, 17-35, 17-30, 17-25, 18-30, 18-25, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19-21, 21-25, 21-25, or 21-23 nucleotides in length, and the antisense strand is independently 15-35, 17-35, 17-30, 25-35, 27-30, 17-23, 17-21, 17-19, 19-25, 19-23, 19- 21, 21-25, 21-25, or 21-23 nucleotides in length. For example, the sense and the antisense strand can be independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. For example, the sense strand is 19, 20, 21, 22 or 23 nucleotides in length and the antisense strand is 21, 22, 23, 24 or 25 nucleotides in length. In some particular embodiments, the sense strand is 20, 21 or 22 nucleotides in length and the antisense strand is 22, 23 or 24 nucleotides in length. For example, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

[0296] The sense strand and antisense strand typically form a double-stranded or duplex region. Without limitations, the duplex region of a dsRNA agent described herein can be 12-35 nucleotide (or base) pairs in length. For example, the duplex region can be between 14-35nucleotide pairs in length, 17-30 nucleotide pairs in length, 17-25 nucleotide pairs in length, 18-25 nucleotide pairs in length, 18-23 nucleotide pairs in length, 25-35 nucleotides in length, 27-35 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotide pairs in length. In some embodiments, the duplex region is 18, 19, 20, 21, 22, 23, 24 or 25 nucleotide pairs in length. For example, the duplex region is 19, 20, 21, 22 or 23 nucleotide pairs in length. In some embodiments, the the duplex region is 20, 21 or 22 nucleotide pairs in length. For example, the dsRNA molecule has a duplex region of 21 base pairs.

[0297] As described herein, the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotide of Formula (IV), Without limitations, the nucleotides of Formula (IV), all can be present in one strand. The nucleotide of Formula (IV) may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.

[0298] In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides of Formula (IV) described herein. The nucleotide of Formula (IV) described herein can be present at any position of the sense strand. For example, the nucleotide of Formula (IV) described herein can be present at a terminal region of the sense strand. For example, the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3 and4, counting from the 5 ’-end of the sense strand. In another non-limiting example, the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3 and 4, counting from the 3 ’-end of the sense strand. In some embodiments, the nucleotide of Formula (IV) can be present at one or more of positions 18, 19, 20 and 21, counting from 5 ’-end of the sense strand. The nucleotide of Formula (IV) described herein can also be located at a central region of sense strand. For example, the nucleotide of Formula (IV) described herein can be located at one or more of positions 6, 7, 8, 9, 10, 11, 12 and 13, counting from 5 ’-end of the sense strand. In some embodiments, the nucleotide of Formula (IV) is at the 5-terminus of the sense strand.

[0299] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of nucleotides of Formula (IV) described herein. The nucleotide of Formula (IV) described herein can be present at any position of the antisense strand. For example, the nucleotide of Formula (IV) described herein can be present at a terminal region of the antisense strand. For example, the nucleotide of Formula (IV) described herein can be present at one or more of positions 1, 2, 3, 4,5, 6, 7, 8, and 9, counting from the 5’-end of the antisense strand. In another non-limiting example, the nucleotide of Formula (IV) described herein nucleotide can be present at one or more of positions 2, 3, 4, 5, 6, 7, and 8, counting from the 3 ’-end of the antisense strand. In someembodiments, the nucleotide of Formula (IV) described herein nucleotide can be present at one or more of positions 6, 7, 8, and 9, counting from 5 ’-end of the antisense strand.

[0300] In some embodiments, the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 1, 2, 3, 4, 5, 6, 7, 8, or 9, counting from the 5 ’-end of the antisense strand. For example, the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 2, 3, 4, 5, 6, 7, or 8, counting from the 5 ’-end of the antisense strand. In some embodiments, the sense strand comprises a nucleotide of Formula (IV) described herein at a position complemenatry to position 6, 7, 8, or 9, counting from the 5 ’-end of the antisense strand.

[0301] As described herein, the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a modified sugar. Accordingly, in some embodiments, the dsRNA agent can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides independently selected from the group consisting of 2’-F, 2-OMe, acyclic nucleotides, locked nucleic acid (LNA), HNA, CeNA, 2 ’-methoxy ethyl, 2’-O-allyl, 2’-C-allyl, 2'-O-N- methylacetamido (2'-0-NMA), a 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-O- aminopropyl (2'-O-AP), and 2'-ara-F. A nucleotide comprising modified sugar can be present anywhere in the dsRNA molecule. For example, a nucleotide comprising a modified sugar can be present in the sense strand or a nucleotide comprising a modified sugar can be present in the antisense strand. When two or more nucleotides comprising a modified sugar are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.

[0302] As described herein, the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro (2’-F) nucleotides. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides. The 2’-fluoro nucleotides can be located anywhere in the sense strand. For example, the sense strand comprises a 2 ’-fluoro nucleotide at position 10, counting from 5 ’-end of the sense strand. In some embodiments, the sense strand comprises a 2’-fluoro nucleotide at position 10, counting from 5’- end of the sense strand and the sense strand further comprises a 2’-fluoro nucleotide at position 8, 9, 11 or 12, counting from 5’-end of the sense strand. For example, the sense strand comprises a 2 ’-fluoro nucleotide at positions 9 10, counting from 5 ’-end of the sense strand. In another example, the sense strand comprises a 2’-fluoro nucleotide at positions 10 and 11, counting from 5 ’-end of the sense strand. In some embodiments, the sense strand comprises a 2 ’-fluoro nucleotide at positions 9, 10 and 11, counting from 5 ’-end of the sense strand. In some other embodiments, the sense strand comprises a 2’-fluoro nucleotide at positions 8, 9 and 10, counting from 5’-end ofthe sense strand. In yet some other embodiments, the sense strand comprises a 2’-fluoro nucleotide at positions 10, 11 and 12, counting from 5 ’-end of the sense strand.

[0303] In some embodiments, the antisense comprises 2 ’-fluoro nucleotides at positions 7, 10 and 11 from the 5’-end. In some other embodiments, the sense strand comprises 2’-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5’-end of the sense strand (e.g., when the sense strand is 21-23 nucleotides in length). In some embodiments, the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand or the first paired nucleotide at the 5 ’end of the antisense strand. In some other embodiments, the sense strand comprises 2’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 13 of the antisense strand, counting from the 5 ’-end of the antisense strand, or the first paired nucleotide at the 5 ’end of the antisense strand. For example, the sense strand can comprise 2’-fluoro nucleotides at positions 7, 8, and 9, counting from the 5 ’-end of the sense strand, when the sense strand is 19 nucleotides in length; or positions 8, 9, and 10, counting from the 5 ’-end of the sense strand, when the sense strand is 20 nucleotides in length; or positions 9, 10, and 11 counting from the 5 ’-end of the sense strand, when the sense strand is 21 nucleotides in length.

[0304] In some other embodiments, the sense strand comprises 2 ’-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand or the first paired nucleotide at the 5 ’end of the antisense stran . For example, the sense strand can comprise 2’-fluoro nucleotides at positions 5, 7, 8, and 9, counting from the 5 ’-end of the sense strand, when the sense strand is 19 nucleotides in length; or positions 6, 8, 9, and 10, counting from the 5 ’-end of the sense strand, when the sense strand is 20 nucleotides in length; or positions 7, 9, 10, and 11 counting from the 5’-end of the sense strand, when the sense strand is 21 nucleotides in length.

[0305] In some embodiments, the sense strand comprises a block of two, three or four 2’- fluoro nucleotides. For example, the sense strand can comprises a block of four 2’-fluoro nucleotides, such as at positions 9, 10, 11, and 12; or 8, 9, 10, and 11, when the sense strand is 21- 23 nucleotides in length (e.g., 21 nucleotides in length).

[0306] In some embodiments, the sense strand does not comprise a 2’-fluoro nucleotide in position opposite or complimentary to a thermally destabilizing modification of the duplex in the antisense strand.

[0307] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-fluoro nucleotides. The 2’-fluoro nucleotides can be located anywhere in the antisense strand. For example, the antisense strand can comprise a 2’-fluoro nucleotide at position 14, counting from 5 ’-end of the antisense strand. In some embodiments, the antisense comprises 2 ’-fluoro nucleotidesat positions 2 and 14, counting from the 5 ’-end of the antisense strand. In some embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 6, 14 and 16 from the 5 ’-end. In some other embodiments, the antisense comprises 2’- fluoro nucleotides at positions 2, 4, and 14 counting from the 5 ’-end of the antisense strand. In some other embodiments, the antisense comprises 2’-fluoro nucleotides at positions 2, 4, 14 and 16 counting counting from the 5 ’-end of the antisense strand. In still some embodiments, the antisense comprises 2’ -fluoro nucleotides at positions 2, 6, 8, 9, 14 and 16 counting from the 5’- end. of the antisense strand In still some embodiments, the antisense comprises 2 ’-fluoro nucleotides at positions 2, 4, 8, 9, 14 and 16 counting from the 5 ’-end of the antisense strand.

[0308] In some embodiments, the antisense strand comprises at least one 2’-fluoro nucleotide adjacent to a destabilizing modification. For example, the 2’-fluoro nucleotide can be the nucleotide at the 5 ’ -end or the 3 ’ -end of a destabilizing modification, i. e. , at position - 1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2 ’-fluoro nucleotide at each of the 5 ’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises at least two 2’-fluoro nucleotides at the 3 ’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

[0309] In some embodiments, both the sense and the antisense strands comprise at least one 2 ’-fluoro nucleotide. The 2 ’-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2’-fluoro modification can occur on every nucleotide on the sense strand and / or antisense strand; each 2’-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2’- fluoro modifications in an alternating pattern. The alternating pattern of the 2’-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2’-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2’-fluoro modifications on the antisense strand.

[0310] As described herein, the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides. Without limitations, the 2’-OMe nucleotides all can be present in one strand. The 2’-OMe nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.

[0311] In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’- OMe nucleotides. The 2’-OMe nucleotides can be located anywhere in the sense strand. In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-OMe nucleotides. The 2’-OMe nucleotides can be located anywhere in the antisense strand.

[0312] As described herein, the dsRNA molecule described herein can comprise at least one, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more 2’-deoxy, e.g., 2’-H ribose nucleotides. For example, the dsRNA can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 2’-deoxy, e.g., 2’-H nucleotides. The 2’-deoxy nucleotide may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand.

[0313] As described herein, the dsRNA can comprise 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 a central region of the sense strand and / or the antisense strand. For example, at least one of the sense stand and the antisense can comprise 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 modification in positions 5-17, e.g., positions 6-16, positions 6-15, positions 6-14, positions 6-13, positions 6-12, positions 7-15, positions 7-14, positions 7-13, positions, 7-12, positions 8-16, positions 8-15, positions 8-14, positions 8-13, positions 8-12, positions 9-16, positions 9-15, positions 9-14, positions 9-13, positions 9-12, positions 10-16, positions 10-15, positions 10-14, positions 10-13 or positions 10-12, counting from the 5 ’-end of the sense strand or the antisense strand.

[0314] In some embodiments, the antisense strand comprises 1, 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides. For example, antisense strand can comprise 2, 3, 4, 5 or 6 of 2’-deoxy nucleotides. The 2’-deoxy nucleotides can be located anywhere in the antisense strand. For example, the antisense strand comprises a 2 ’-deoxy nucleotide at 1, 2, 3, 4, 5 or 6 of positions 2, 5, 7, 12, 14 and 16, counting from 5 ’-end of the antisense strand. In one non-limiting example, the antisense strand comprises a 2 ’-deoxy nucleotide at 1, 2, 3 or 4 of positions 2, 5, 7, and 12, counting from 5 ’-end of the antisense strand.

[0315] In some embodiments, the antisense comprises a 2 ’-deoxy nucleotide at positions 5 and 7, counting from 5’-end of the antisense strand. For example, the antisense strand comprises a 2’- deoxy nucleotide at positions 5, 7 and 12, counting from 5 ’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5 and 7, counting from 5’-end of the antisense strand. For example, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7 and 12, counting from 5’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at positions 2, 5, 7, 12 and 14, counting, from 5’-end of the antisense strand. For example, the antisense strand comprises a 2’- deoxy nucleotide at positions 2, 5, 7, 12, 14 and 16, counting from 5’-end of the antisense strand

[0316] In some embodiments, the antisense comprises a 2’-deoxy nucleotide at position 2 or 12, counting from 5’-end of the antisense strand. For example, the antisense comprises a 2’-deoxy nucleotide at position 12, counting from 5 ’-end of the antisense strand.

[0317] In some embodiments, the dsRNA comprises at least three 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at position 11 of the sense strand, counting from 5 ’-end of the sense strand.

[0318] In some embodiments, the dsRNA comprises at least five 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2, 12 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5’- end of the sense strand.

[0319] In some embodiments, the dsRNA comprises at least seven 2’-deoxy modifications, wherein the 2 ’-deoxy modifications are at positions 2, 5, 7, 12 and 14 of the antisense strand, counting from 5 ’-end of the antisense strand, and at positions 9 and 11 of the sense strand, counting from 5 ’-end of the sense strand.

[0320] In some embodiments, the antisense strand comprises at least five 2’-deoxy modifications at positions 2, 5, 7, 12 and 14, counting from 5 ’-end of the antisense strand.

[0321] In one non-limiting example, the sense strand does not comprise a 2 ’-deoxy nucleotide at position 11, counting from 5 ’-end of the sense strand.

[0322] In some embodiments, the dsRNA can comprise one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides comprising a non-natural nucleobase

[0323] A nucleotide comprising a non-natural nucleobase can be present anywhere in the dsRNA molecule. For example, a nucleotide comprising a non-natural nucleobase can be present in the sense strand or a nucleotide comprising a non-natural nucleobase can be present in the antisense strand. When two or more nucleotides comprising a non-natural nucleobase are present in the dsRNA molecule, they can all be in the sense strand, antisense strand or both in the sense and antisense strands.

[0324] The dsRNA molecule described herein can further comprise at least one phosphorothioate or methylphosphonate intemucleoside linkage. The phosphorothioate or methylphosphonate intemucleoside linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the intemucleoside linkage modification may occur on every nucleotide on the sense strand and / or antisense strand; each intemucleoside linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both intemucleoside linkage modifications in an alternating pattern. The alternating pattern of the intemucleoside linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the intemucleoside linkage modification on the sense strand may have a shiftrelative to the alternating pattern of the intemucleoside linkage modification on the antisense strand.

[0325] In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate intemucleoside linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate intemucleoside linkage between the two nucleotides. Intemucleoside linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate intemucleoside linkage, and optionally, there may be additional phosphorothioate or methylphosphonate intemucleoside linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate intemucleoside linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3 ’-end of the antisense strand.

[0326] In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0327] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0328] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0329] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0330] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0331] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0332] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0333] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3, 4, 5 or 6 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0334] In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate intemucleoside linkages separated by 1, 2, 3 or 4 phosphate intemucleoside linkages, wherein one of the phosphorothioate or methylphosphonate intemucleoside linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate intemucleoside linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

[0335] In some embodiments, the dsRNA molecule described herein further comprises one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the termini position(s) of the sense and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate or methylphosphonate intemucleoside linkage at one end or both ends of the sense and / or antisense strand.

[0336] In some embodiments, the dsRNA molecule described herein comprises one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the internal region of the duplex of each of the sense and / or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked through phosphorothioate methylphosphonate intemucleoside linkage at position 8-16 of the duplex region counting from the 5 ’-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate intemucleoside linkage modification within 1-10 of the termini position(s).

[0337] In some embodiments, the dsRNA molecule described herein further comprises one to five phosphorothioate or methylphosphonate intemucleoside linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification(s) within the last 3 positions of the sense strand (counting from the 5 ’-end), and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification at positions 1 and 2 and one to five phosphorothioate or methylphosphonate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).

[0338] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and onephosphorothioate or methylphosphonate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate intemucleoside linkage modifications within the last six the last six positions of the antisense strand (counting from the 5 ’-end).

[0339] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0340] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and two phosphorothioate intemucleoside linkage modifications within the last four positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0341] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and two phosphorothioate intemucleoside linkage modifications within the last four positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).

[0342] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last four positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0343] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 and one within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modification at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).

[0344] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification within position 1-5 (counting from the 5’- end) of the sense strand, and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).

[0345] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 (counting from the 5’- end) of the sense strand, and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0346] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and one phosphorothioate intemucleoside linkage modification within the last six positions of the antisense strand (counting from the 5 ’-end).

[0347] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0348] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications within position 1-5 and one phosphorothioate intemucleoside linkage modification within the last six positions of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications within the last six positions of the antisense strand (counting from the 5 ’-end).

[0349] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 20 and 21 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5 ’-end).

[0350] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioateintemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 20 and 21 the antisense strand (counting from the 5 ’-end).

[0351] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 21 and 22 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one phosphorothioate intemucleoside linkage modification at position 21 of the antisense strand (counting from the 5 ’-end).

[0352] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 21 and 22 the antisense strand (counting from the 5 ’-end).

[0353] In some embodiments, the dsRNA molecule described herein further comprises two phosphorothioate intemucleoside linkage modifications at position 1 and 2, and two phosphorothioate intemucleoside linkage modifications at position 22 and 23 of the sense strand (counting from the 5 ’-end), and one phosphorothioate intemucleoside linkage modification at positions 1 and one phosphorothioate intemucleoside linkage modification at position 21 of the antisense strand (counting from the 5 ’-end).

[0354] In some embodiments, the dsRNA molecule described herein further comprises one phosphorothioate intemucleoside linkage modification at position 1, and one phosphorothioate intemucleoside linkage modification at position 21 of the sense strand (counting from the 5 ’-end), and two phosphorothioate intemucleoside linkage modifications at positions 1 and 2 and two phosphorothioate intemucleoside linkage modifications at positions 22 and 23 the antisense strand (counting from the 5 ’-end).

[0355] In some embodiments, the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand.

[0356] In some embodiments, the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisensestrand. For example, the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand.

[0357] In some embodiments, the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’ end of the antisense strand. For example, the antisense strand comprises phosphorothioate linkages between nucleotides n and n-1, and between nucleotides n-1 and n-2, where n is length of the antisense strand, i.e, number of nucleotides in the antisense strand. In other words, the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.

[0358] In some embodiments, the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’ -end of the antisense strand and at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the antisense strand. For example, the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand and between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.

[0359] In some embodiments, the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5 ’-end of the antisense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the antisense strand.

[0360] In some embodiments, the sense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 5’ end of the sense strand and the antisense strand comprises at least two phosphorothioate intemucleoside linkages between the first five nucleotides counting from the 3 ’-end of the antisense strand. For example, the sense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 5 ’-end of the sense strand, and the antisense strand comprises phosphorothioate linkages between nucleotides 1 and 2, and between nucleotides 2 and 3, counting from 3 ’-end of the antisense strand.

[0361] In some embodiments, dsRNA molecule described herein comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 intemucleotidic linkages in the Sp configuration. In some embodiments, acommon pattern of backbone chiral centers comprises at least 6 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 intemucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 intemucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern ofbackbone chiral centers comprises no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 intemucleotidic linkages in the Sp configuration, and no more than 8 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 intemucleotidic linkages in the Sp configuration, and no more than 7 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 intemucleotidic linkages in the Sp configuration, and no more than 6 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 intemucleotidic linkages in the Sp configuration, and no more than 5 intemucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 intemucleotidic linkages in the Sp configuration, and no more than 4 intemucleotidic linkages which are not chiral. In some embodiments, the intemucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the intemucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

[0362] In some embodiments, dsRNA molecule described herein comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each intemucleotidic linkage of the block is Rp. In some embodiments, a 5 ’-block is an Rp block. In some embodiments, a 3 ’-block is an Rp block. In some embodiments, a block is an Sp block in that each intemucleotidic linkage of the block is Sp. In some embodiments, a 5 ’-block is an Sp block. In some embodiments, a 3 ’-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rpblocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each intemucleotidic linkage in a natural phosphate linkage.

[0363] In some embodiments, dsRNA molecule described herein comprises a 5 ’-block is an Sp block wherein each sugar moiety comprises a 2 ’-fluoro modification. In some embodiments, a 5 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 5’- block is an Sp block wherein each of intemucleoside linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 5’-block comprises 4 or more nucleoside units. In some embodiments, a 5 ’-block comprises 5 or more nucleoside units. In some embodiments, a 5 ’-block comprises 6 or more nucleoside units. In some embodiments, a 5 ’-block comprises 7 or more nucleoside units. In some embodiments, a 3 ’-block is an Sp block wherein each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a modified intemucleotidic linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block is an Sp block wherein each of intemucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2’-fluoro modification. In some embodiments, a 3 ’-block comprises 4 or more nucleoside units. In some embodiments, a 3 ’-block comprises 5 or more nucleoside units. In some embodiments, a 3 ’-block comprises 6 or more nucleoside units. In some embodiments, a 3 ’-block comprises 7 or more nucleoside units.

[0364] In some embodiments, dsRNA molecule described herein comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of intemucleotidic linkage, e.g., natural phosphate linkage, modified intemucleotidic linkage, Rp chiral intemucleotidic linkage, Sp chiral intemucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

[0365] Various publications describe multimeric siRNA which can all be used with the oligonucleotide and dsRNA of the invention. Such publications include W02007 / 091269, USPatent No. 7858769, W02010 / 141511, W02007 / 117686, W02009 / 014887 and WO2011 / 031520 which are hereby incorporated by their entirely.

[0366] In some embodiments, the dsRNA molecule described herein comprises one or more overhang regions and / or capping groups of dsRNA molecule at the 3 ’-end, or 5 ’-end or both ends of a strand. The overhang can be 1-10 nucleotides in length. For example, the overhang can be 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, the overhang is 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target sequence or it can be complementary to the gene sequences being targeted or it can be the other sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

[0367] In some embodiments, the nucleotides in the overhang region of the dsRNA molecule described herein can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2’-Fluoro 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl- 5 -methyluridine, 2 ’-O-methoxy ethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA, SNA, hGNA, hhGNA, mGNA, TNA, h’GNA, and any combinations thereof. For example, dTdT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be other sequence.

[0368] The 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule described herein may be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3 ’-end of the sense strand, antisense strand or both strands. In some embodiments, this 3 ’-overhang is present in the antisense strand. In some embodiments, this 3 ’-overhang is present in the sense strand.

[0369] The dsRNA molecule described herein may comprise only a single overhang, which can strengthen the interference activity of the dsRNA, without affecting its overall stability. For example, the single-stranded overhang is located at the 3 '-terminal end of the sense strand or, alternatively, at the 3'-terminal end of the antisense strand. The dsRNA can also have a blunt end, located at the 5 ’-end of the antisense strand (or the 3 ’-end of the sense strand) or vice versa.

[0370] Generally, the antisense strand of the dsRNA has a nucleotide overhang at the 3 ’-end, and the 5 ’-end is blunt. While not bound by theory, the asymmetric blunt end at the 5 ’-end of theantisense strand and 3 ’-end overhang of the antisense strand favor the guide strand loading into RISC process. For example, the single overhang is at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides in length. In some embodiments, the dsRNA has a 2 nucleotide overhang on the 3 ’-end of the antisense strand and a blunt end at the 5 ’-end of the antisense strand.

[0371] The dsRNA described herein can comprise one or more modified nucleotides. For example, every nucleotide in the sense strand and antisense strand of the dsRNA molecule can be modified. Each nucleotide can be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens and / or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar; replacement of the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

[0372] As nucleic acids are polymers of subunits, many of the modifications occur at aposition which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3’ or 5’ terminal position, may only occur in a central region, may only occur at a non-terminal region, or may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of a RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5’ end or ends can be phosphorylated.

[0373] It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5’ or 3’ overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3’ or 5’ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2’ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2 ’-deoxy-2’ -fluoro (2’-F) or 2’-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

[0374] In some embodiments, the dsRNA molecule described herein comprises modifications of an alternating pattern, particular in the Bl, B2, B3, Bl’, B2’, B3’, B4’ regions. The term “alternating motif’ or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “AB AB AB AB AB AB... ,” “AABB AABB AABB ... ,” “AAB AABAAB AAB ... ,” “AAAB AAABAAAB ... ,”“AAABBBAAABBB. .. ,” or “AB CAB CAB CAB C... ,” etc.

[0375] The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “AB AB AB...”, “AC AC AC...” “BDBDBD...” or “CDCDCD... ,” etc.

[0376] In some embodiments, the dsRNA molecule described herein comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “AB AB AB” from 5 ’ -3 ’ of the strand and the alternating motif in the antisense strand may start with “BAB AB A” from 3’-5’of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABB AABB” from 5 ’-3’ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3’-5’of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

[0377] In some embodiments of any one of the aspects described herein, the oligonucleotides described herein or at least one e.g., both strand of a dsRNA described herein are 5’ phosphorylated or include a phosphoryl analog at the 5’ prime terminus. 5'-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5'- monophosphate ((HO)2(O)P-O-5'); 5 '-diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'); 5 '-triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-guanosine cap (7-methylated or non-methylated) (7m-G-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5'-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5'-(HO)(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); 5 '-monothiophosphate (phosphorothioate; (HO)2(S)P-O-5'); 5 '-monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), 5'-phosphorothiolate ((HO)2(O)P-S-5'); any additional combination of oxygen / sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5'- alpha-thiotriphosphate, 5 '-gamma-thiotriphosphate, etc.), 5 '-phosphorami dates ((HO)2(O)P-NH-5', (HO)(NH2)(O)P-O-5'), 5'-alkylphosphonates (e.g., RP(0H)(0)-0-5'-, R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc.), 5'-alkenylphosphonates (i.e. vinyl, substituted vinyl, e.g., OH)2(O)P-5'- CH= or (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates (e.g., R(0H)(0)P-0-5', R=alkylether, e.g., methoxymethyl (MeOCH2-), ethoxymethyl, etc.) Other exemplary 5 ’-modifications include where Z is optionally substituted alkyl at least once, e.g., ((HO)2(X)P-O[-(CH2)a-O-P(X)(OH)-O]b- 5', ((HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5', ((HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5'; dialkyl terminal phosphates and phosphate mimics: H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O- P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(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 a and b are each independently 1-10. Other embodiments, include replacement of oxygen and / or sulfur with BH3, BHV and / or Se.

[0378] In some embodiments of any one of the aspects described herein, the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-vinylphosphonate group. For example, the oligonucleotide or at least one (e.g., both) strand of a dsRNA described herein comprises a 5’-E-vinyl or at least one (e.g., both) strand of a dsRNA described herein phosphonate group. In some other non-limiting example, the oligonucleotide comprises a 5’-Z- vinylphosphonate group.

[0379] In one example, the 5 ’-modification can be placed in the antisense strand of a double- stranded nucleic acid, e.g., dsRNA molecule. For example, the antisense comprises a 5’-E- vinylphosphonate. In some other non-limiting example, the antisense strand comprises a 5’-Z- vinylphosphonate group.

[0380] In another example, the 5 ’-terminal nucleotide of the antisense strand comprises a 5’- cyclopropylphosphonate group, that is, a group of the formula or a salt thereof,that is connected to the 4’-C of the 5 ’-end nucleotide.

[0381] In another example, the 5 ’-terminal nucleotide of the antisense strand comprises a group of the formula wherein each Rppis independently hydrogen or a Cl-6alkyl(e.g., methyl) or a salt thereof, connected to the 4’-C of the 5’-terminal nucleotide. In one example, the group is of the formulaor a salt thereof.

[0382] In some embodiments, the sense strand comprises a 5 ’-morpholino, a 5’- dimethylamino, a 5 ’-deoxy, an inverted abasic, or an inverted abasic locked nucleic acid modification at the 5 ’-end. In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphodiester linkage to an abasic nucleotide. In some embodiments, , the 5’- terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphorothioate linkage to an abasic nucleotide.

[0383] In some embodiments, the sense strand comprises an inverted abasic acid modification at the 3 ’-end. In some embodiments, the 3 ’-terminal nucleotide of the sense strand comprises a 3’- >3’ phosphodiester linkage to an abasic nucleotide. In some embodiments, he 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.

[0384] In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphodiester linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphodiester linkage to an abasic nucleotide.

[0385] In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphorothioate linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphodiester linkage to an abasic nucleotide. In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5 ’->5’ phosphodiester linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.

[0386] In some embodiments, the 5 ’-terminal nucleotide of the sense strand comprises a 5’- >5’ phosphorothioate linkage to an abasic nucleotide; and the 3 ’-terminal nucleotide of the sense strand comprises a 3 ’->3’ phosphorothioate linkage to an abasic nucleotide.

[0387] In each of the preceding embodiments of a terminal modification by an abasic nucleotide, the abasic nucleotide may be optionally substituted, for example, by any of the modifications or ligand described herein. The dsRNA agents of the invention can comprise thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2- 9 of the 5 ’-end of the antisense strand or positions 2-9 counting from the first paired nucleotide)of the duplex region at the 5 ’-end of the antisense strand) to reduce or inhibit off-target gene silencing. Without wishing to be bound by a theory, dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, countingfrom the 5’ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5’ region of the antisense strand. In some embodiments, thermally destabilizing modification of the duplex is located in positions 2-9, or preferably positions 4-8, from the 5 ’-end of the antisense strand. In some further embodiments, the thermally destabilizing modification of the duplex is located at position 5, 6, 7 or 8 from the 5’-end of the antisense strand.

[0388] In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5 ’-end of the antisense strand.

[0389] In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand and / or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.

[0390] In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14 and 16 from the 5 ’-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14 and 16 from the 5’-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14 and 16 from the 5 ’-end.

[0391] In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5 ’-end or the 3 ’-end of the destabilizing modification, i.e., at position -1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5’-end and the 3 ’-end of the destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.

[0392] In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3 ’-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification. In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10 and 11 from the 5 ’-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10 and 11 from the 5 ’-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12 and 15 of the antisense strand, counting from the 5 ’-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary 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 a block of two, three or four stabilizing modifications.

[0393] In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

[0394] It is noted a thermally stabilizing modification can replace a 2’-fluoro nucleotide in the sense and / or antisense strand. For example, a 2’-fluoro nucleotide at positions 8, 9, 10, 11 and / or 12, counting from 5 ’-end, of the sense strand, can be replaced with a thermally stabilizing modification. Similarly, a 2’-fluoro nucleotide at position 14, counting from 5’-end, of the antisense strand, can be replaced with a thermally stabilizing modification.

[0395] For the dsRNA molecules to be more effective in vivo, the antisense strand must have some metabolic stability. In other words, for the dsRNA molecules to be more effective in vivo, some amount of the antisense stand may need to be present in vivo after a period time after administration. Accordingly, in some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 5 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 6 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 7 after in vivo administration. In some embodiments, at least 40%, for example at least 45%, at least 50%, at least 55%, at least 60%., atleast 65%, at least 70%, at least 75%, or at least 80% of the antisense strand of the dsRNA is present in vivo, for example in mouse liver, at day 8 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 9 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 10 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 11 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 12 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 13 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 14 after in vivo administration. In some embodiments, 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 the dsRNA is present in vivo, for example in mouse liver, at day 15 after in vivo administration.Uses of oligonucleotides and dsRNAs

[0396] In some embodiments of any one of the aspects, the oligonucleotide described herein or the antisense strand of the dsRNA molecule described herein comprises a nucleotide sequence substantially complementary to a target nucleic acid, e.g., a target gene or mRNA.

[0397] Accordingly, in another aspect, the disclosure is directed to a use of an oligonucleotide and / or dsRNA molecule described herein for inhibiting expression of a target gene. In some embodiments, the present invention further relates to a use of an oligonucleotide and / or dsRNA molecule described herein for inhibiting expression of a target gene in vitro.

[0398] In another aspect, the disclosure is directed to a use of an oligonucleotide and / or dsRNA molecule described herein for use in inhibiting expression of a target gene in a subject. The subject may be any animal, such as a mammal, e.g., a mouse, a rat, a sheep, a cattle, a dog, a cat, or a human

[0399] In some embodiments, the oligonucleotide and / or dsRNA molecule described herein is administered in buffer.

[0400] In some embodiments, oligonucleotide and / or dsRNA molecule described herein described herein can be formulated for administration to a subject. A formulated oligonucleotide and / or dsRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and / or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the siRNA is in an aqueous phase, e.g., in a solution that includes water.

[0401] The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the siRNA composition is formulated in a manner that is compatible with the intended method of administration, as described herein. For example, in particular embodiments the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly.

[0402] A oligonucleotide and / or dsRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes an oligonucleotide and / or dsRNA, e.g., a protein that complexes with oligonucleotide and / or dsRNA. Still other agents include chelating agents, e.g., EDTA (e.g., to remove divalent cations such as Mg2+), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

[0403] In some embodiments, the oligonucleotide and / or dsRNA preparation includes another dsRNA compound, e.g., a second dsRNA that can mediate RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different siRNA species. Such dsRNAs can mediate RNAi with respect to a similar number of different genes.

[0404] In some embodiments, the oligonucleotide and / or dsRNA preparation includes at least a second therapeutic agent (e.g., an agent other than a RNA or a DNA). For example, a oligonucleotide and / or dsRNA composition for the treatment of a viral disease, e.g., HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a dsRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

[0405] Exemplary formulations which can be used for administering the oligonucleotide and / or dsRNA according to the present invention are discussed below.

[0406] Liposomes. A oligonucleotide and / or dsRNA preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide and / or dsRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the oligonucleotide and / or dsRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the oligonucleotide and / or dsRNA are delivered into the cell where the dsRNA can specifically bind to a target RNA and can mediate RNAi. In some embodiments, the liposomes are also specifically targeted, e.g., to direct the oligonucleotide and / or dsRNA to particular cell types.

[0407] A liposome containing oligonucleotide and / or dsRNA can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The dsRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the siRNA and condense around the dsRNA to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of oligonucleotide and / or dsRNA.

[0408] If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favor condensation.

[0409] Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate apolynucleotide / cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96 / 37194. Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413- 7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, etal. Biochim. Biophys. Acta 557:9, 1979; Szoka, etal. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim.Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984, which are incorporated by reference in their entirety. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986, which is incorporated by reference in its entirety). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta T15A69, 1984, which is incorporated by reference in its entirety). These methods are readily adapted to packaging oligonucleotide and / or dsRNA preparations into liposomes.

[0410] Liposomes that are pH-sensitive or negatively-charged entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274, which is incorporated by reference in its entirety).

[0411] One major type of liposomal composition includes phospholipids other than naturally- derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and / or phosphatidylcholine and / or cholesterol.

[0412] Examples of other methods to introduce liposomes into cells in vitro and include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94 / 00569; WO 93 / 24640; WO 91 / 16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90: 11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

[0413] In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver siRNAs to macrophages.

[0414] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated siRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0415] A positively charged synthetic cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of siRNA (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA, which are incorporated by reference in their entirety).

[0416] A DOTMA analogue, l,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, l,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moi eties are linked by ester, rather than ether linkages.

[0417] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5 -carboxy spermylgly cine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5 -carboxy spermyl-ami de (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

[0418] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating poly lysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. etal., Biochim. Biophys. Acta 1065:8, 1991, which is incorporated by reference in its entirety). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containingcompositions. Other commercially available cationic lipid products include DMRIE and DMRIE- HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0419] Liposomal formulations are particularly suited for topical administration. Liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer siRNA, into the skin. In some implementations, liposomes are used for delivering siRNA to epidermal cells and also to enhance the penetration of siRNA into dermal tissues, e.g., into skin. Lor example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Lould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987, which are incorporated by reference in their entirety).

[0420] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with dsRNA descreibed herein are useful for treating a dermatological disorder.

[0421] Liposomes that include oligonucleotide and / or dsRNA described herein can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. Lor example, transfersomes are a type of deformable liposomes. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include oligonucleotide and / or dsRNA described herein can be delivered, for example, subcutaneously by infection in order to deliver dsRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self- repairing, and can frequently reach their targets without fragmenting, and often self-loading.

[0422] Other formulations amenable to the present invention are described in United States provisional application serial nos. 61 / 018,616, fded January 2, 2008; 61 / 018,611, fded January 2, 2008; 61 / 039,748, filed March 26, 2008; 61 / 047,087, filed April 22, 2008 and 61 / 051,528, filed May 8, 2008. PCT application no PCT / US2007 / 080331, filed October 3, 2007 also describes formulations that are amenable to the present invention.

[0423] Surfactants. The oligonucleotide and / or dsRNA compositions can include a surfactant. In some embodiments, the dsRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile / lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0424] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0425] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0426] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0427] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0428] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0429] Micelles and other Membranous Formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0430] A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the oligonucleotide and / or dsRNA composition, an alkali metal Cs to C22 alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyl oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

[0431] In one method, a first micellar composition is prepared which contains the oligonucleotide and / or dsRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the dsRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

[0432] Phenol and / or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and / or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

[0433] For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it isnecessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

[0434] Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen- containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1, 1,2 tetrafluoroethane) may be used.

[0435] The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

[0436] Particles. In some embodiments, dsRNA preparations can be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.Pharmaceutical compositions

[0437] The oligonucleotide and / or dsRNA described herein can be formulated for pharmaceutical use. The present invention further relates to a pharmaceutical composition comprising the oligonucleotide and / or dsRNA described herein. Pharmaceutically acceptable compositions comprise a therapeutically-effective amount of one or more of the dsRNA molecules in any of the preceding embodiments, taken alone or formulated together with one or more pharmaceutically acceptable carriers (additives), excipient and / or diluents.

[0438] The pharmaceutical compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; (3) topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin; (4) intravaginally or intrarectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; or (8) nasally. Delivery using subcutaneous or intravenous methods can be particularly advantageous.

[0439] The phrase “therapeutically-effective amount” as used herein means that amount of a compound, material, or composition comprising a dsRNA molecule described herein which isI l leffective for producing some desired therapeutic effect in at least a sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0440] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0441] The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as com starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0442] The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 0.1 per cent to about ninety -nine percent of activeingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.

[0443] In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, celluloses, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound of the present invention. In certain embodiments, an aforementioned formulation renders orally bioavailable a compound of the present invention.

[0444] Methods of preparing these formulations or compositions include the step of bringing into association an oligonucleotide and / or dsRNA with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0445] In some cases, in order to prolong the effect of a drug, it is desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.

[0446] The oligonucleotide and / or dsRNA described herein may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.

[0447] The term “treatment” is intended to encompass therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans, and other mammals such as equines, cattle, swine and sheep; and poultry and pets in general.

[0448] The oligonucleotide and / or dsRNA described herein or a pharmaceutical composition comprising an oligonucleotide and / or dsRNA described herein can be administered to a subject using different routes of delivery. A composition that includes an oligonucleotide and / or dsRNA described herein described herein can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

[0449] The oligonucleotide and / or dsRNA described herein may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0450] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the oligonucleotide and / or dsRNA described herein in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the oligonucleotide and / or dsRNA described herein and mechanically introducing the oligonucleotide and / or dsRNA described herein.

[0451] In one aspect, provided herein is a method of administering an oligonucleotide and / or dsRNA described herein, to a subject (e.g., a human subject). In another aspect, the present invention relates to an oligonucleotide and / or dsRNA described herein for use in inhibiting expression of a target gene in a subject. The method or the medical use includes administering a unit dose of the oligonucleotide and / or dsRNA described herein. In some embodiments, the unit dose is less than 10 mg per kg of body weight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of body weight, and less than 200 nmole of RNA agent (e.g., about 4.4 x 1016copies) per kg of body weight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of oligonucleotide and / or dsRNA described herein per kg of body weight.

[0452] The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target gene. The unit dose, for example, can be administered by injection (e.g., intravenous, subcutaneous or intramuscular), an inhaled dose, or a topical application. In some embodiments dosages may be less than 10, 5, 2, 1, or 0.1 mg / kg of body weight.

[0453] In some embodiments, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

[0454] In some embodiments, the effective dose is administered with other traditional therapeutic modalities.

[0455] In some embodiments, a subject is administered an initial dose and one or more maintenance doses. The maintenance dose or doses can be the same or lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 pg to 15 mg / kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are, for example, administered no more than once every 2, 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In certain embodiments the dosage may be delivered nomore than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

[0456] The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracistemal or intracapsular), or reservoir may be advisable.

[0457] In some embodiments, the composition includes a plurality of dsRNA molecule species. In another embodiment, the dsRNA molecule species has sequences that are non- overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of dsRNA molecule species is specific for different naturally occurring target genes. In another embodiment, the dsRNA molecule is allele specific.

[0458] The oligonucleotide and / or dsRNA described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

[0459] In some embodiments, the administration of the oligonucleotide and / or dsRNA composition described herein is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

[0460] The invention provides methods, compositions, and kits, for rectal administration or delivery of oligonucleotide and / or dsRNA composition described herein.Methods of inhibiting expression of a target gene

[0461] Aspects of the disclosure also relate to methods for inhibiting the expression of a target gene. The method comprises administering to the subject in an amount sufficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the first strand is complementary to a target gene; and / or (ii) an oligonucleotide described herein, wherein the oligonucleotide is complementary to a target gene.

[0462] The present disclosure further relates to a use of an oligonucleotide and / or dsRNA molecule ...

Claims

CLAIMSWhat is claimed is1. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein each strand is independently 15 to 35 nucleotides in length, and wherein the first or second strand comprises at least one nucleoside of Formula (IV):Formula (IV) wherein:B’ is an optionally substituted nucleobase;XMis CH2, O, NRNor S, where RNis aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars;R43is a bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2- soalkynyl, optionally substituted C1-30 alkoxy (e.g., methoxy), alkoxy alkyl (e.g., 2- methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group;R45represents a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker covalently bonded a solid support, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-l-yl, piperidin-l-yl, or pyrrolidin-l-yl), halogen, alkoxy alkyl (e.g., 2-methoxy ethyl), alkoxy alkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (H0)(HS)(S)P-0-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (H0)(NH2)(0)P-0- 5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rpis optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(OH)(O)P-O- 5', RP1is alkoxyalkyl, e.g., methoxymethyl (CFPOMe) or ethoxymethyl ], (H0)2(X)P- O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O- P(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', or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., -CH =CH-XP, Xpis a phosphonate group) or C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), whereinX is O or S; a and b are each independently 1-10, provided that only one of R43and R45is a solid support or linker covalently bonded to a solid support; and each R8and R9is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl, and provided that,(i) when R43is not a bond to an intemucleotide linkage to a subsequent nucleotide, thenR45is a bond to an intemucleotide linkage to a preceding nucleotide; and(ii) when R45is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R43is a bond to an intemucleotide linkage to a preceding nucleotide.

2. The double-stranded nucleic acid of claim 1, wherein XMis CH2.

3. The double-stranded nucleic acid of claim 1, wherein XMis O.

4. The double-stranded nucleic acid of claim 1, wherein XMis S.

5. The double-stranded nucleic acid of any one of claims 1-4, wherein R43is bond to an intemucleotide linkage to a subsequent nucleotide, hydrogen, hydroxyl, protected hydroxyl, a nitrogen protecting group, a linker, a 3’-oligonuclotide capping group, aligand, a linker covalently bonded to one or more ligands, a lipid, or a linker covalently bonded to one or more lipids.

6. The double-stranded nucleic acid of claim 6, wherein R43is a bond to an intemucleotide linkage to a subsequent nucleotide.

7. The double-stranded nucleic acid of claim 6, wherein R43is either (i) hydrogen or a nitrogen protecting group; or (ii) hydroxyl or a protected hydroxyl.

8. The double-stranded nucleic acid of any one of claims 1-7, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, phosphate mimic, or a bond to an intemucleotide linkage to a preceding nucleotide, or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

9. The double-stranded nucleic acid of claim 8, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded a solid support, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma-thiotriphosphate, or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

10. The double-stranded nucleic acid of claim 9, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide.

11. The double-stranded nucleic acid of claim 9, wherein R45is optionally substituted C1-30 alkoxy or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

12. The double-stranded nucleic acid of claim 9, wherein R45is hydroxyl.

13. The double-stranded nucleic acid of any one of claims 1-12, wherein B’ is unmodified nucleobase (e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 deaza purine, optionally modified at the N7 position.

14. The double-stranded nucleic acid of any one of claims 1-13, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one ribonucleotide.

15. The double-stranded nucleic acid of any one of claims 1-14, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one 2’-deoxyribonucleotide.

16. The double-stranded nucleic acid of any one of claims 1-15, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a modified or non-natural nucleobase in addition to the nucleotide of Formula (IV).

17. The double-stranded nucleic acid of any one of claims 1-16, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a modified ribose sugar in addition to the nucleotide of Formula (IV).

18. The double-stranded nucleic acid of any one of claims 1-17, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide comprising a group other than H or OH at the 2 ’-position of the ribose sugar in addition to the nucleotide of Formula (IV).

19. The double-stranded nucleic acid of any one of claims 1-18, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a 2’-F ribose in addition to the nucleotide of Formula (IV).

20. The double-stranded nucleic acid of any one of claims 1-19, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide with a 2’- OMe ribose in addition to the nucleotide of Formula (IV).

21. The double-stranded nucleic acid of any one of claims 1-20, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one nucleotide comprising a moiety other than a ribose sugar in addition to the nucleotide of Formula (IV).

22. The double-stranded nucleic acid of any one of claims 1-21, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one modified intemucleotide linkage.

23. The double-stranded nucleic acid of any one of claims 1-22, wherein at least one of the first or second strand comprises at least one ligand.

24. The double-stranded nucleic acid of claim 23, wherein the strand comprising the nucleotide of Formula (IV) comprises at least one ligand.

25. The double-stranded nucleic acid of any one of claims 1-24, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the strand comprising the nucleotide of Formula (IV).

26. The oligonucleotide of any one of claims 1-25, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the strand comprising the nucleotide of Formula (IV)., and wherein the nucleoside of Formula (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphodiester intemucleotide linkage.

27. The double-stranded nucleic acid of any one of claims 1-26, wherein the nucleoside of Formula (IV) is present at 3’-end of the strand comprising the nucleotide of Formula (IV)., and wherein the nucleoside of Formula (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphorothioate internucleotide linkage.

28. The double-stranded nucleic acid of any one of claims 1-27, wherein the first and second strand are independently 18 to 25 nucleotides in length.

29. The double-stranded nucleic acid of any one of claims 1-28, wherein the first and second strand form a double-stranded or duplex region of 17 to 25 basepairs.

30. The double-stranded nucleic acid any one of claims 1-29, wherein double-stranded nucleic acid is capable of inducing RNA interference.

31. The double-stranded nucleic acid of any one of claims 1-30, wherein one or both strands have a 1 – 5 nucleotide overhang on its respective 5’-end or 3’-end.

32. The double-stranded nucleic acid of any one of claims 1-31, wherein only one strand has a 2 nucleotide overhang on its 5’-end or 3’-end.

33. The double-stranded nucleic acid of any one of claims 1-32, wherein only one strand has a 2 nucleotide overhand on its 3’-end.

34. An oligonucleotide comprising at least one nucleoside of Formula (IV):Formula (IV) , wherein: B’ is an optionally substituted nucleobase; XMis CH2, O, NRNor S, where RNis aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars; R43is a bond to an internucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30alkoxy (e.g., methoxy), alkoxyalkyl (e.g., 2-methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, -0-C4-3oalkyl-ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), or a nitrogen protecting group;R45represents a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker covalently bonded a solid support, hydrogen, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkyl, optionally substituted C2-3oalkenyl, optionally substituted C2-3oalkynyl, optionally substituted C1-30 alkoxy, optionally substituted 3-8 membered heterocyclyl (e.g., morpholin-l-yl, piperidin-l-yl, or pyrrolidin-l-yl), halogen, alkoxy alkyl (e.g., 2-methoxy ethyl), alkoxy alkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -0-C4-3oalkyl- ON(CH2R8)(CH2R9), -0-C4-3oalkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH-5', (H0)(NH2)(0)P-0- 5'), alkylphosphonates [(Rp)(0H)(0)P-0-5', Rpis optionally substituted C1-30 alkyl, e.g., methyl, ethyl, isopropyl, or propyl)], alkyletherphosphonates [(Rpl)(OH)(O)P-O- 5', RP1is alkoxyalkyl, e.g., methoxymethyl (CFPOMe) or ethoxymethyl ], (H0)2(X)P- O[-(CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., H0[-(CH2)a-0-P(X)(0H)-0]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O- P(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', or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group (e.g., -CH=CH-XP, Xpis a phosphonate group), C3-6 cycloalkylphosphonate (e.g., cyclopropylphosphonate), whereinX is O or S; a and b are each independently 1-10, provided that only one of R43and R45is a solid support or linker covalently bonded to a solid support; and each R8and R9is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted Ci-3oalkynyl, and provided that,(i) when R43is not a bond to an intemucleotide linkage to a subsequent nucleotide, thenR45is a bond to an intemucleotide linkage to a preceding nucleotide; and(ii) when R45is not a bond to an intemucleotide linkage to a subsequent nucleotide, then R43is a bond to an intemucleotide linkage to a preceding nucleotide.

35. The oligonucleotide of claim 34, wherein R43is bond to an intemucleotide linkage to a subsequent nucleotide, a solid support, a linker, a linker covalently bonded a solid support, a 3’-oligonuclotide capping group, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently bonded to one or more lipids, hydrogen, hydroxyl, protected hydroxyl, or a nitrogen protecting group.

36. The oligonucleotide of claim 35, wherein R43is a bond to an intemucleotide linkage to a subsequent nucleotide.

37. The oligonucleotide of claim 35, wherein R43is a solid support, or a linker (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) covalently bonded to a solid support, hydrogen or a nitrogen protecting group.

38. The oligonucleotide of any one of claims 34-37, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha- thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate, phosphate mimic, or a bond to an intemucleotide linkage to a preceding nucleotide or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

39. The oligonucleotide of claim 38, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide, a solid support, a linker, a linker covalently bonded a solid support, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, or gamma- thiotriphosphate, or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group..

40. The oligonucleotide of claim 39, wherein R45is a bond to an intemucleotide linkage to a preceding nucleotide.

41. The oligonucleotide of claim 39, wherein R45is hydroxyl, protected hydroxyl, or optionally substituted C1-30 alkoxy, or R45taken together with the carbon to which it is attached form a vinylphosphonate (VP) group.

42. The oligonucleotide of any one of claims 34-41, wherein XMis CH2.

43. The oligonucleotide of any one of claims 34-41, wherein XMis O.

44. The oligonucleotide of any one of claims 34-41, wherein XMis S.

45. The oligonucleotide of any one of claims 34-44, wherein B’ is unmodified nucleobase(e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 deaza purine, optionally modified at the N7 position.

46. The oligonucleotide of any one of claims 34-45, wherein the oligonucleotide comprises from 3 to 50 nucleotides.

47. The oligonucleotide of any one of claims 34-46, wherein the oligonucleotide comprises at least one ribonucleotide.

48. The oligonucleotide of any one of claims 34-47, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.

49. The oligonucleotide of any one of claims 34-48, wherein the oligonucleotide comprises at least one nucleotide with a modified or non-natural nucleobase in addition to the nucleotide of Formula (IV).

50. The oligonucleotide of any one of claims 34-49, wherein the oligonucleotide comprises at least one nucleotide with a modified ribose sugar in addition to the nucleotide of Formula (IV).

51. The oligonucleotide of any one of claims 34-50, wherein the oligonucleotide comprises at least one nucleotide comprising a group other than H or OH at the 2’-position of the ribose sugar in addition to the nucleotide of Formula (IV).

52. The oligonucleotide of any one of claims 34-51, wherein the oligonucleotide comprises at least one nucleotide with a 2’-F ribose in addition to the nucleotide of Formula (IV).

53. The oligonucleotide of any one of claims 34-52, wherein the oligonucleotide comprises at least one nucleotide with a 2’-0Me ribose in addition to the nucleotide of Formula (IV).

54. The oligonucleotide of any one of claims 34-53, wherein the oligonucleotide comprises at least one nucleotide comprising a moiety other than a ribose sugar in addition to the nucleotide of Formula (IV).

55. The oligonucleotide of any one of claims 34-54, wherein the oligonucleotide comprises at least one modified intemucleotide linkage.

56. The oligonucleotide of any one of claims 34-55, wherein the oligonucleotide is attached to a solid support.

57. The oligonucleotide of any one of claims 34-56, wherein oligonucleotide comprises at least one ligand.

58. The oligonucleotide of any one of claims 34-57, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group.

59. The oligonucleotide of any one of claims 34-58, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide.

60. The oligonucleotide of any one of claims 34-59, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, and wherein the nucleoside of Formule (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphodiester intemucleotide linkage.

61. The oligonucleotide of any one of claims 34-60, wherein the nucleoside of Formula (IV) is present at 3 ’-end of the oligonucleotide, and wherein the nucleoside of Formule (IV) is linked to the preceding nucleoside (i.e., nucleoside 5’ to it) by a phosphorothioate intemucleotide linkage.

62. A method of reducing the expression of a target gene in a subject, comprising administering to the subject either:(i) a double-stranded RNA according to any one of claims 1-33 or 92-120, wherein the first strand or the second strand is complementary to a target gene; or(ii) an oligonucleotide according to any one of claims 34-61 or 86-91, wherein the oligonucleotide is complementary to a target gene.

63. A compound of Formula (III):Formula (III) wherein:B’ is an optionally substituted nucleobase;XMis CH2, O, NRNor S, where RNis aliphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars;R33is hydrogen, hydroxy, protected hydroxy, nitrogen protecting group, phosphate group, a reactive phosphorous group, a solid support, a linker, a linker covalently bonded (e.g.,-C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, optionally substituted C1-30alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), or -O-C4-30alkyl- ON(CH2R8)(CH2R9); R35is hydroxy, protected hydroxy, phosphate group, a reactive phosphorous group, optionally substituted C1-30alkyl, optionally substituted C2-30alkenyl, optionally substituted C2-30alkynyl, optionally substituted C1-30 alkoxy, halogen, alkoxyalkyl (e.g., methoxyethyl), alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, -O-C4-30alkyl-ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), monophosphate ((HO)2(O)P-O-5'), diphosphate ((HO)2(O)P-O-P(HO)(O)-O-5'), triphosphate ((HO)2(O)P-O-(HO)(O)P-O-P(HO)(O)-O-5'); monothiophosphate (phosphorothioate, (HO)2(S)P-O-5'), monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P-O-5'), phosphorothiolate ((HO)2(O)P-S-5'); alpha-thiotriphosphate; beta-thiotriphosphate; gamma-thiotriphosphate; phosphoramidates ((HO)2(O)P-NH- 5', (HO)(NH2)(O)P-O-5'), alkylphosphonates (R(OH)(O)P-O-5', R=alkyl, e.g., methyl, ethyl, isopropyl, propyl, etc…), alkyletherphosphonates (R(OH)(O)P-O-5', R=alkylether, e.g., methoxymethyl (CH2OMe), ethoxymethyl, etc…), (HO)2(X)P-O[- (CH2)a-O-P(X)(OH)-O]b- 5' or (HO)2(X)P-O[-(CH2)a-P(X)(OH)-O]b- 5' or (HO)2(X)P-[-(CH2)a-O-P(X)(OH)-O]b- 5', where X is O, S or optionally substituted alkyl, and dialkyl terminal phosphates and phosphate mimics (e.g., HO[-(CH2)a-O- P(X)(OH)-O]b- 5' , H2N[-(CH2)a-O-P(X)(OH)-O]b- 5', H[-(CH2)a-O-P(X)(OH)-O]b- 5', Me2N[-(CH2)a-O-P(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 1-10), provided that only one of R33and R35is a reactive phosphorous group; or R35taken together with the carbon to which it is attached form a vinylphosphonate (VP) group or C3-6cycloalkylphosphonate (e.g., cyclopropylphosphonate), a vinylphosphonate (VP) group; and each R8and R9is independently H, a targeting ligand (e.g., GalNac), a pharmacokinetics modifier, optionally substituted C1-30alkyl, optionally substituted C1-30alkenyl, or optionally substituted C1-30alkynyl.

64. The compound of claim 63, wherein R33is H, a linker, a ligand, a linker covalently bonded to one or more ligands, a lipid, a linker covalently attached to one or more lipids, or a nitrogen protecting group.

65. The compound of claim 63, wherein R33is (i) a H or nitrogen protecting group or (ii) hydroxy or protected hydroxy.

66. The compound of any one of claims 63-65, wherein R35is a reactive phosphorous group, linker, solid support, a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or 5’-O- C(O)CH2CH2C(O)-) to a solid support, hydroxyl, protected hydroxyl, optionally substituted C1-30 alkoxy, monophosphate, diphosphate, triphosphate, monothiophosphate (phosphorothioate), monodithiophosphate, phosphorothiolate, alpha-thiotriphosphate, beta-thiotriphosphate, gamma-thiotriphosphate, phosphoramidate, alkylphosphonate, alkyletherphosphonate, dialkyl terminal phosphate or phosphate mimic, or R35taken together with the carbon to which it is attached form a vinylphosphonate group.

67. The compound of any one of claims 63-66, wherein R35is a reactive phosphorous group, linker, solid support, a linker covalently bonded to a solid support, hydroxyl, or a protected hydroxyl.

68. The compound of any one of claims 63-67, wherein R35is reactive phosphorous group, solid support, or a linker covalently bonded to a solid support.

69. The compound of any one of claims 63-68, wherein R35is -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)70. The compound of any one of claims 63-69, wherein XMis CH2.

71. The compound of any one of claims 63-69, wherein XMis O.

72. The compound of any one of claims 63-69, wherein XMis S.

73. The compound of claim 63, wherein:XMis CH2;R33is H or nitrogen protecting group (e.g., trityl); andR35is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support, a linker covalently bonded (e.g., - C(O)CH2CH2C(O)-) to a solid support, hydroxyl, or a protected hydroxyl.

74. The compound of claim 63, wherein:XMis O;R33is H or nitrogen protecting group (e.g., trityl); andR35is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support a linker covalently bonded (e.g., - C(O)CH2CH2C(O)-) to a solid support, hydroxyl, or a protected hydroxyl.

75. The compound of claim 63, wherein:XMis S;R33is H or nitrogen protecting group (e.g., trityl); andR35is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support a linker covalently bonded (e.g., - C(O)CH2CH2C(O)-) to a solid support, hydroxyl, or a protected hydroxyl.

76. The compound of claim 63, wherein:XMis CH2;R35is hydroxyl or a protected hydroxyl; andR33is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.

77. The compound of claim 76, wherein:XMis CH2;R35is hydroxyl or a protected hydroxyl; andR33is a solid support or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.

78. The compound of claim 63, wherein:XMis O;R35is hydroxyl or protected hydroxyl; andR33is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.

79. The compound of claim 78, wherein:XMis O;R35is hydroxyl or protected hydroxyl; andR33is a solid support, or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.

80. The compound of claim 63, wherein:XMis S;R35is hydroxyl or protected hydroxyl; andR33is a reactive phosphorous group (e.g., -P(XD)(N(RP2)2)-RP4, where XDis O or S; each RP2is independently an optionally substituted Ci-Cealkyl (e.g., methyl); and RP4is halogen (e.g., Cl)), solid support, or a linker covalently bonded (e.g., - C(O)CH2CH2C(O)- or -OC(O)CH2CH2C(O)-) to a solid support.

81. The compound of claim 80, wherein:XMis S;R35is hydroxyl or protected hydroxyl; andR33is a solid support, or a linker covalently bonded (e.g., -C(O)CH2CH2C(O)- or - OC(O)CH2CH2C(O)-) to a solid support.

82. The compound of any one of claims 63-81, B’ is an unmodified nucleobase (e.g., adenine, cytosine, guanine, thymine or uracil), a pyrimidine modified at the C4 position, a pyrimidine modified at the C5 position, a purine modified at the N2 position, a purine modified at the N6 position, a purine modified at the C6 position or a N-7 deaza purine, optionally modified at the N7 position.

83. The compound of any one of claims 63-82, wherein B and B’ are independently adenine, cytosine, guanine, thymine, uracil,selected from 1 to 10; and R1is independently liphatic and aromatic alkyl, alkylester, alkylamine, branched alkylamine, dimethylamino alkyl, alkylether, alkylthioether, heteroaromatic alkyl, allyl, vinyl, alkyl groups functionalized with disulfide, oxime, ketone, acetal, hemiacetal, cleavable peptides, cleavable sugars.A compound selected from the group consisting of:(where @ is a solid support, e.g., CPG),(where X is O, CH2. NR or S, and R is a protecting group or an85. An oligonucleotide prepared using a compound of any one of claims 63-84.

86. The oligonucleotide of any one of claims 34-61, wherein the nucleotide of Formula (IV) is at one of positions 2-9, counting from the 5 ’end of the oligonucleotide.

87. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5- 7 or at one of positions 6-8, counting from the 5 ’end of the oligonucleotide.

88. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 5, counting from the 5 ’end of the oligonucleotide.

89. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 6, counting from the 5 ’end of the oligonucleotide.

90. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 7, counting from the 5 ’end of the oligonucleotide.

91. The oligonucleotide of claim 86, wherein the nucleotide of Formula (IV) is at position 8, counting from the 5 ’end of the oligonucleotide.

92. A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantially complementary to the first strand, wherein one of first or second oligonucleotide strand is an oligonucleotide of any one of claims 34-61 or 86- 91.

93. The double-stranded nucleic acid of claim 92, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the sense strand.

94. The double-stranded nucleic acid of claim 93, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide) from the 5 ’-end of the antisense strand).

95. The double-stranded nucleic acid of claim 94, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

96. The double-stranded nucleic acid of claim 94, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

97. The double-stranded nucleic acid of claim 94, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

98. The double-stranded nucleic acid of claim 94, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

99. The double-stranded nucleic acid of claim 94, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

100. The double-stranded nucleic acid of claim 92, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the antisense strand.

101. The double-stranded nucleic acid of claim 100, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-9, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

102. The double-stranded nucleic acid of claim 101, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-8, at one of positions 2-7, at one of positions3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

103. The double-stranded nucleic acid of claim 101, wherein the nucleotide of Formula (IV) is in the antisense strand at position 5, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

104. The double-stranded nucleic acid of claim 101, wherein the nucleotide of Formula (IV) is in the antisense strand at position 6, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

105. The double-stranded nucleic acid of claim 101, wherein the nucleotide of Formula (IV) is in the antisense strand at position 7, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

106. The double-stranded nucleic acid of claim 101, wherein the nucleotide of Formula (IV) is in the antisense strand at position 8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotidefrom the 5 ’-end of the antisense strand.

107. The double-stranded nucleic acid of any one of claims 1-33, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the sense strand.

108. The double-stranded nucleic acid of claim 107, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-9 of the antisense strand, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

109. The double-stranded nucleic acid of claim 108, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) one of positions 2-8, one of positions 2-7, one of positions 3-8, one of positions 3-7, one of positions 4-8, one of positions 4-7, one of positions 5-8, one of positions 5-7 or one of positions 6-8 of the antisense strand, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

110. The double-stranded nucleic acid of claim 107, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 5 of the antisense strand, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

111. The double-stranded nucleic acid of claim 107, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 6 of the antisense strand, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

112. The double-stranded nucleic acid of claim 107, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 7 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

113. The double-stranded nucleic acid of claim 107, wherein the nucleotide of Formula (IV) is in the sense strand at a position that is opposite to (i.e., forms a base pair with) position 8 of the antisense strand, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

114. The double-stranded nucleic acid of any one of claims 1-33, wherein the double-stranded nucleic acid is an siRNA and the strand comprising the nucleotide of Formula (IV) is the antisense strand.

115. The double-stranded nucleic acid of claim 114, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-9, counting from the 5 ’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

116. The double-stranded nucleic acid of claim 115, wherein the nucleotide of Formula (IV) is in the antisense strand at one of positions 2-8, at one of positions 2-7, at one of positions 3-8, at one of positions 3-7, at one of positions 4-8, at one of positions 4-7, at one of positions 5-8, at one of positions 5-7 or at one of positions 6-8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand).

117. The double-stranded nucleic acid of claim 115, wherein the nucleotide of Formula (IV) is in the antisense strand at position 5, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

118. The double-stranded nucleic acid of claim 115, wherein the nucleotide of Formula (IV) is in the antisense strand at position 6, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

119. The double-stranded nucleic acid of claim 115, wherein the nucleotide of Formula (IV) is in the antisense strand at position 7, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

120. The double-stranded nucleic acid of claim 115, wherein the nucleotide of Formula (IV) is in the antisense strand at position 8, counting from the 5’end of the antisense strand (or counting from the first paired nucleotide from the 5 ’-end of the antisense strand.

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