5'-modified monomers, oligonucleotides and double-stranded rnas

Abstract

The technology described herein relates to 5'-modified nucleosides, nucleotides, oligonucleotides and double-stranded RNAs, e.g., siRNAs, and kits comprising them and methods of their use for inhibiting target genes.

Description

Aty. Dkt. No.051058-000104WOPT 5’-MODIFIED MONOMERS, OLIGONUCLEOTIDES AND DOUBLE-STRANDED RNAS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under § 119(e) of U.S. Provisional Application No. 63 / 521,256 filed June 15, 2023, contents of al of which are incorporated herein by reference in their entireties. TECHNICAL FIELD

[0002] The technology described herein relates generaly to 5’-modified nucleosides, nucleotides, oligonucleotides and double-stranded RNAs, e.g., siRNAs, compositions and kits comprising them and methods of their use for inhibiting target genes. BACKGROUND

[0003] There remains a need in the art for oligonucleotides and siRNAs having improved activity and / or pharmacodynamics. The present disclosure addresses some of these needs. SUMMARY

[0004] In one aspect, provided herein is a compound of Formula I,or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil or 5-methyluracil); X is O or S; each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); one of R2 and R3 is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); the other of R2 and R3 is -OR30, wherein: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

[0005] In some embodiments, X is O. In some other embodiments, X is S.

[0006] In compounds of Formula I, each RV is independently hydrogen or a hydroxyl protecting group. In some embodiments, each RV is hydrogen. In some embodiments, each RV is a hydroxyl protecting group, such as pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM) or ethyl (CH- 3CH2-).

[0007] In some compounds described herein, R3is -OR30. For example, R3is -OR30, and R30 is a reactive phosphorous group. Exemplary reactive phosphorous groups are described herein below and include, but are not limited to, phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Thus, in some embodiments, R3 is -OR30, and R30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionaly R30 is a phosphoramidite. Accordingly, in some compounds, R30 is -P(ORP1)N(RP2), -P(SRP1)N(RP2), -P P1 P2 2 2 (O)(OR )N(R)2, - P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SRP1)N(RP2) P1 P1 2 2 2, -P(O)(OR)H, -P(S)(OR )H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, such as isopropyl) or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.

[0008] In some embodiments, R3is -OR30, and R30 is -P(ORP1)N(RP2) 3 2. For example, Ris - OR30, and R30 is -P(ORP1)N(RP2), and substituted with cyano or -SC(O) In some embodiments, R3is -OR30, and , and wher P1 2 e R is –CH2CH2CN.

[0009] In some embodiments, 3-P(ORP1)N(RP2)2, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R3is -OR30, and R30 is - P(ORP1)N(RP2), and where P2 2 each R is isopropyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0010] In some preferred embodiments, R3is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C alkyl substituted with cyano or -SC(O)Ph, an P2 1-6 d each R is independently methyl, ethyl, propyl, or isopropyl. For example, R3is -OR30, and R30 is -P(ORP1)N(RP2) P1 2, and where R is – CHCHCN, and each P2 2 2 R is isopropyl.

[0011] In some embodiments, R3is -OR30, and R30 is a phosphoramidite group such as 3'-[(2- cyanoethyl)-(N,N-disopropyl)]-phosphoramidite or 3'-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite).

[0012] In another example, R3is -OR30, and R30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group). Some exemplary hydroxyl protecting group for R30 of R3include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS.

[0013] In yet another example, R3is -OR30, and R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R30 is a bond to an oligonucleotide. When R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide, R3can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide. It is noted that the internucleotide linkage between the compound or nucleoside of Formula I and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below.

[0014] In some embodiments, R3is hydrogen or halogen (e.g., F, Br, Cl or I). For example, R3 is H or F.

[0015] In some compounds of Formula I, R3is -OR20, where R20 is optionaly substituted C1- alkyl. For example, R3is -OR20, where R20 is methyl, ethyl, or propyl 3 6 . In some embodiments, R is -OR20, where R20 is methyl.

[0016] In some compounds of Formula I, R3is -OR20, where R20 is optionaly substituted C2- alkenyl. For example, R3is -O 20 20 6 R, where R is vinyl or alyl.In some compounds of Formula I, R3is -OR20, where R20 is optionaly substituted C 3 20 20 2-6alkynyl. For example, Ris -OR, where R is acetylenyl, propargyl, or 5-hexyn-1-yl.

[0017] In some compounds of Formula I, R3is -OR20, where R20 is C1-6alkoxyC1-6alkyl. For example, R3is -OR20, where R202-methoxyethyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0018] Preferably, R3is -OR30 and R2 is hydrogen, halogen, or -OR20. For example, R3is - OR30 and R2 is hydrogen, halogen, or -OR20, and where R30 is a reactive phosphorous group, hydroxyl protecting group, a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

[0019] In some embodiments, R3is -OR30, R2 is hydrogen or halogen (e.g., F, Br, Cl or I), and where R30 is a reactive phosphorous group, hydroxyl protecting group, a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R2 is H or F.

[0020] In some compounds of Formula I, R2is -OR20, where R20 is optionaly substituted C1- 6alkyl. In one example, R20 is C1-6alkyl substituted with one, two, or three substituents selected independently from the group consisting of halogen, -OR22, -N(R22) 22 22 2, -SR , -C(O)OR, - C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxyprop- 2-yl). In another example, R20 is C1-6alkyl substituted with one or two substituents selected independently from the group consisting of halogen, -OR22, -N(R22)2, -SR22, -C(O)OR22, - C(O)N(R22)2, wherein R22 is hydrogen or C1-3alkyl (e.g., 2,2,2-trifluoroethyl, 1,3-dimethoxyprop- 2-yl).

[0021] In another example, R2is -OR20, where R20 is methyl, ethyl, or propyl. In some embodiments, R2is -OR20, where R20 is methyl.

[0022] In some compounds of Formula I, R2is -OR20, where R20 is C1-6alkoxyC1-6alkyl. For example, R2is -OR20, where R20is 2-methoxyethyl.

[0023] In some compounds of Formula I, R2 is -OR20, where R20 is N-(C1- 6alkyl)aminocarbonylC1-6alkyl. For example, R2is -OR20, where R20is 2-(N-methylamino)-2- oxoethyl.

[0024] In another example, R2is -OR30, and R30 is hydrogen or a hydroxyl protecting group (e.g., a silyl based hydroxyl protecting group). Some exemplary hydroxyl protecting group for R30 of R2include, but are not limited to, t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS.

[0025] In some compounds described herein, R2is -OR30. For example, R2is -OR30, and R30 is a reactive phosphorous group. Exemplary reactive phosphorous groups are described herein below and include, but are not limited to, phosphoramidite, H-phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Thus, in some embodiments, R2 is -OR30, and R30 is a reactive phosphorous group selected from phosphoramidite, H-phosphonate, alkyl-phosphonate, and phosphate triester, optionaly R30 is a phosphoramidite. Accordingly, in some compounds, R30 is -P(ORP1)N(RP2), -P(SRP1 P2 P1 P2 2 )N(R )2, -P(O)(OR )N(R)2, - 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, such as isopropyl) or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.

[0027] In some embodiments, R2is -OR30, and R30 is -P(ORP1)N(RP2)2, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R2is -OR30, and R30 is - P(ORP1)N(RP2) P2 2, and where each R is isopropyl.

[0028] In some preferred embodiments, R2is -OR30, and R30 is -P(ORP1)N(RP2)2, and where RP1 is C1-6alkyl substituted with cyano or -SC(O)Ph, and each RP2 is independently methyl, ethyl, propyl, or isopropyl. For example, R2is -OR30, and R30 is -P(ORP1)N(RP2) P1 2, and where R is – CHC P2 2 H2CN, and each R is isopropyl.

[0029] In some embodiments, R2is -OR30, and R30 is a phosphoramidite group such as 2’-[(2- cyanoethyl)-(N,N-disopropyl)]-phosphoramidite or 2’-[(ß-thiobenzoylethyl)-(1-pyrrolidinyl)]- thiophosphoramidite).

[0030] In yet another example, R2is -OR30, and R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R30 is a bond to an oligonucleotide. When R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide, R2can be connected to the 5’- hydroxyl of the nucleoside or nucleotide or the 5’-hydroxyl at the 5’-terminal of the oligonucleotide. It is noted that the internucleotide linkage between the compound or nucleoside of Formula I and the nucleoside, nucleotide, or oligonucleotide it is linked to can be an unmodified internucleotide linkage (i.e., phosphodiester) or a modified internucleotide linkage (e.g., phosphorothioate, MMI or imidp, preferably the modified internucleotide linkage is phosphorothioate). Exemplary modified internucleotide linkages are described herein below. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0031] In compounds of Formula I, B is an optionaly modified natural or non-natural nucleobase. For example, B is uracil, adenine, cytosine, 5-methylcytosine, guanine, or thymine (i.e., 5-methyluracil). In some embodiments, B is a modified or protected nucleobase. For example, B is a protected nucleobase comprising at least one amine or hydroxyl protecting group. In some embodiments, B is adenine, cytosine, 5-methylcytosine, or guanine comprising at least one amine protecting group. Exemplary modified, unmodified natural and non-natural nucleobase are described herein below.

[0032] In some compounds of Formula I, X is O; R2is hydrogen, F, or -OR20, where R20is hydrogen, optionaly substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such as C1-6alkoxyC1- 6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6alkyl)aminocarbonylC1-6alkyl (e.g.2-(N- methylamino)-2-oxoethyl); and R3 is -OR30, where R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.

[0033] In some compounds of Formula I, X is O; R2is hydrogen, F, or -OR20, where R20is hydrogen, optionaly substituted C1-6alkyl (e.g., methyl, ethyl or propyl), such asC1-6alkoxyC1- 6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N- methylamino)-2-oxoethyl); and R3 is -OR30, where R30 is reactive phosphorous group (e.g., a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester). In some further embodiments of this, the reactive phosphorous group is: -P(ORP1)N(RP2) P1 P2 2, -P(SR )N(R )2,- P(O)(ORP1)N(RP2) P1 P2 P3 P2 P1 P2 P1 2, -P(S)(OR )N(R )2, -P(R )N(R )2, -P(O)(SR )N(R)2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3 (e.g., - P(ORP1)N(RP2)2), wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2-C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C P1 2-C10alkenyl, or optionaly substituted C2-C10alkynyl); each R is independently an optionaly substituted C1-6alkyl (e.g., C1-6alkyl substituted with cyano or - SC(O)Ph, such as 2-cyanoethyl); and each RP2 is independently optionaly substituted C1-6alkyl (e.g., methyl, ethyl, propyl, or isopropyl, preferably isopropyl), or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.

[0034] In some compounds of Formula I, X is O; R2is hydrogen, F, or -OR20, where R20is hydrogen, optionaly substituted C1-6alkyl (e.g., methyl, ethyl or propyl, preferably methyl), such as C1-6alkoxyC1-6alkyl (e.g., methoxy, 2-methoxyethyl) or N-(C1-6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl); and R3 is -OR30, where R30 is -P(ORP1)N(RP2)2, and where RP1 is –CHCH P2 2 2CN, and each R is isopropyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0035] In some preferred embodiments, X is O; R2is hydrogen, F, or -OR20, where R20 is hydrogen, methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, or 2-(N-methylamino)-2-oxoethyl; and R3 is -OR30, where R30 is -P(ORP1)N(RP2) P1 P2 2, where R is –CH2CH2CN, and each R is isopropyl.

[0036] In some embodiments, the compound is of the formula,or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil); X is O or S; each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl; one of R2and R3is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); and the other of R2and R3is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

[0037] In some embodiments, the compound is of the formula,or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil); X is O or S; 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl; one of R2and R3is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); and the other of R2and R3is -OR30, wherein: R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

[0038]

[0039] In some embodiments, the compound is of the formula,salt thereof.

[0040] In some embodiments, the compound is of thesalt thereof.

[0041] In some embodiments, the compound is of thesalt thereof.

[0042] In some embodiments, R3is -OR30. For example, the compound is of formula, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT ,or a salt thereof, wherein: R3 is -OR30, wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide; B is an optionaly modified nucleobase (e.g., uracil); X is O or S; each RP is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl); and R2 is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl);

[0043] In certain embodiments, B is uracil or thymine. In certain embodiments, RV is hydrogen. In certain embodiments, RV is a hydroxyl protecting group (e.g. ethyl or pivaloyloxymethyl).

[0044] In some embodiments, R3 is -OR30, R30 is -P(ORP1)N(RP2)2, and X is O. For example, the compound is of formula, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT ,or a salt thereof, wherein: R3’ is -OR30, wherein: R30 is -P(ORP1)N(RP2)2, wherein: each RP1 is optionaly substituted C1-6alkyl, (e.g., -CH2CH2CN); each RP2 is independently optionaly substituted C1-6alkyl (e.g., isopropyl); X is O; B is an optionaly modified nucleobase (e.g., uracil); each RP is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl); and R2’ is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2- oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2- 6alkynyl (e.g., propargyl).

[0045] In certain embodiments, B is uracil or thymine. In certain embodiments, RV is hydrogen. In certain embodiments, RV is a hydroxyl protecting group (e.g. ethyl or pivaloyloxymethyl). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT X

[0046] In some compounds of Formula (I),portion of the compound isX, RV, R2 and R3 are as defined herein for Formula (I). For example, the compound is of structure:.

[0047] In some embodiments, the compound is of the formula,, or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil); X is O or S; 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl); R2is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); andrepresents the remainder of an oligonucleotide (e.g., the antisense strand of a double-stranded RNA).

[0048] In the preceding, the oxygen atom that is ilustrated linking the 5’-end of the oligonucleotide to the phosphorous atom is the 5’-oxygen of the 5’-terminal nucleoside of the oligonucleotide.

[0049] In some embodiments, Y is S. In other embodiments, Y is O.

[0050] used to represent an oligonucleotide; such oligonucleotides may be an RNA, a DNA, a single-stranded RNA, such as an antisense oligonucleotide (ASO), the antisense strand of a double-stranded RNA (such as an siRNA), and oligonucleotide derivatives such as phosphorodiamidate morpholino oligomers (PMOs).

[0051] In some embodiments, the compound is of the formula,4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTor a salt thereof, wherein: RV is hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl). In certain embodiments, B is uracil or thymine. In certain embodiments, RV is hydrogen. In certain embodiments, RV is a hydroxyl protecting group (e.g. pivaloyloxymethyl). In some embodiments, Y is S. In other embodiments, Y is O.

[0052] In some embodiments, the compound is selected from the group of compounds shown in Table A: Table A: Some exemplary compounds4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0166] In another aspect, provided herein is an oligonucleotide with a 5’-terminal modification comprising the structure:, wherein: X is O or S; and each RV is independently hydrogen or a hydroxyl protecting group.

[0053] In some embodiments, the 5’-terminal modification comprises the structure:

[0054] In certain embodiments, X is O. In certain embodiments, X is O and each RVis hydrogen. In certain embodiments, X is O and each RVis ethyl. In certain embodiments, X is O and each RVis pivaloyloxymethyl. X

[0055] It is noted that the sugar moiety of the nucleotide comprisingmodification, i.e., the nucleotide at 5’-end of the oligonucleotide (5’-terminal nucleotide) can comprise a 5- or 6- membered ring. For example, the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, alopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof).

[0056] Generaly, themodification replaces a CH2OH group on the sugar moiety of a 5’-terrminal nucleotide of the oligonucleotide. For example, the4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTmodification replaces the 4’-CH2OH group on the furanose ring (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose) or the 5’-CH2OH group on the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, alopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5’-terminal nucleotide of the oligonucleotide.

[0057] In some embodiments, the oligonucleotide comprises at its 5’-end a compound of Formula (I) described herein. For example, the 5’-terminal nucleotide of the oligonucleotide is of the structure:, wherein: each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); B is an optionaly modified nucleobase (e.g., uracil); one of R2and R3is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2- oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2- 6alkynyl (e.g., propargyl); and the other of R2and R3is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0058] In some embodiments, the 5’-terminal nucleotide of the oligonucleotide has the tuone of R2and R3is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0059] In some other embodiments, the 5’-terminal nucleotide of the oligonucleotide has theone of R2and R3is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0060] In some other embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: one of R2and R3is -OR30 (e.g., R3 is 30-OR), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0061] In some other embodiments, the 5’-terminal nucleotide of the oligonucleotide has the structure: one of R2and R3is -OR30 3 30(e.g., R is -OR), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0062] In some other embodiments, the 5’-terminal nucleotide of the oligonucleotide has the, where one of R2and R3is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0063] Generaly, the oligonucleotide described herein comprises at least three nucleotides. For example, the oligonucleotide comprises from 5 to 100, e.g., from 10 to 50 nucleotides. In some embodiments, the oligonucleotide comprises from 15 to 40 nucleotides. For example, the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length. For example, the oligonucleotide is 19, 20, 21, 22, or 23 nucleotides in length. It is noted that the compound of Formula I counts as one nucleotide. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0064] The oligonucleotide described herein can comprise at least one nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications). Exemplary nucleic acid modifications are described herein below, and include, but are not limited to nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and combinations thereof.

[0065] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides.

[0066] In some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermaly destabilizing modification of the duplex. For example, the oligonucleotide comprises a thermaly destabilizing modification at at least one of position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, where the compound of Formula I is at position 1 from the 5’-end of the oligonucleotide; optionaly, the thermaly destabilizing modification is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermaly destabilizing modification is located at position 7, counting from the 5’- end of the oligonucleotide.

[0067] In some embodiments, the oligonucleotide comprises least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides. For example, the oligonucleotide comprises 2, 3, 4, 5, or 62’-F nucleotides, optionaly, the oligonucleotide comprises 3, 4, 5 or 62’-F nucleotides. In some embodiments, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula I is at position 1 from the 5’-end of the oligonucleotide. For example, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, preferably, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide. It is noted that when more than one 2’-F nucleotide is present in the oligonucleotide, each 2’-F nucleotide is an independently selected nucleotide.

[0068] The oligonucleotide can also comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides. For example, the oligonucleotide comprises 2, 3, 4, 5, 6, or 7 2’-deoxy nucleotides, optionaly, the oligonucleotide comprises 3, 4, 5 or 62’-deoxy nucleotides. The oligonucleotide can comprise a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula I is at position 1 from the 5’-end of the oligonucleotide. For example, the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide. In some embodiments, the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT counting from the 5’-end of oligonucleotide. For example, the oligonucleotide comprises a 2’- deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of oligonucleotide. For example, thew oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide. It is noted that when more than one 2’-dexy nucleotide is present in the oligonucleotide, each 2’-deoxy nucleotide is an independently selected nucleotide.

[0069] One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) nucleobases in the oligonucleotide can be non-natural or modified nucleobases. For example, the oligonucleotide can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified or protected nucleobases.

[0070] The internucleotide linkages in the oligonucleotide can be independently unmodified (e.g., phosphodiester) or modified (e.g., phosphorothioate). Thus, in some embodiments, the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages. Generaly, the oligonucleotide comprises at least one (e.g., 1, 2, 4, or 5) modified internucleoside linkages (e.g., phosphorothioate) at the first 1-5 positions at one or both ends of the oligonucleotide. For example, the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a modified oligonucleotide linkage (e.g., (e.g., phosphorothioate) between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide.

[0071] In some embodiments, the oligonucleotide is covalently linked to a support, e.g., a solid support.

[0072] In yet another aspect, provided herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand is substantialy complementary to the antisense strand, and wherein one of the sense or the antisense strand is an oligonucleotide described herein, i.e., an oligonucleotide with a 5’-terminal modification comprising the structure:wherein: X is O or S; and each RV is independently hydrogen or a hydroxyl protecting group.

[0073] Preferably, the antisense strand comprises the above 5’-terminal modification. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0074] In stil another aspect, provided herein is a method for reducing the expression of a target gene in a subject. The method comprises administering to the subject either: (i) a double- stranded RNA described herein, where the antisense strand is substantialy complementary to a target gene; or (i) an oligonucleotide described herein, where the oligonucleotide is substantialy complementary to a target gene.

[0075] In another aspect, provided herein is a pharmaceutical composition comprising an oligonucleotide or dsRNA molecule described herein alone or in combination with a pharmaceuticaly acceptable carrier or excipient.

[0076] In yet another aspect, provided herein is a cel comprising an oligonucleotide or dsRNA molecule described herein.

[0077] In stil another aspect, provided herein is a gene silencing kit comprising an oligonucleotide or dsRNA molecule described herein.

[0078] Also, provided herein is a method for silencing a target gene, in a cel. The method comprises a step of introducing: (i) a dsRNA molecule described herein into the cel, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantialy complementary to a nucleotide sequence of the target gene; and / or (i) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantialy complementary to a nucleotide sequence of the target gene.

[0079] 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 dsRNA molecule described herein, where one of the strands, e.g., the antisense of the dsRNA comprises a nucleotide sequence substantialy complementary to a nucleotide sequence of the target gene; and / or (i) an oligonucleotide described herein, wherein the oligonucleotide comprises a nucleotide sequence substantialy complementary to a nucleotide sequence of the target gene. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIGS.1A-1F depict synthesis schemes for the synthesis of some exemplary compounds of the disclosure.

[0081] FIG.2 shows RNAi activity of some exemplary dsRNAs targeting mTTR and comprising exemplary compounds of the disclosure.

[0082] FIG.3 depicts SOD1 evaluation of exemplary dsRNA comprising exemplary in rats.

[0083] FIGS.4-7 depict molecular modeling studies. FIG.4, 6-ʹE-VP-RNA sits in a congested space in the case of 6’-E-VP-RNA and it takes the position of the water molecule in the parent structure. FIG.5, spacing is realy tight and the water that is inserted there in the 4F3T (PDB) miR-20a complex with Ago2 is displaced. Otherwise, there is absolutely no room left 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT between the phosphate that is now inserted a bit deeper into the pocket and various basic side chains. The reference structure has been superimposed on al others and is colored in gray. FIG.6, 6-ʹZ-VP modification accommodated wel; it appears like it accommodates beter than the 6-ʹE- VP-isomer which has steric clash with Gln-545 and Tyr-529; and 6-ʹZ-VP maintains beter stacking interaction with Tyr-529 than 5’-Z-VP. FIG.7, the 6’-Z-VP-U modification at AS1 fits wel into the MID binding pocket – it looks beter than the 6’-E-VP case that pushes the terminal phosphate a bit too close to multiple basic residues as wel as Gln-545 and Tyr-529. DETAILED DESCRIPTION

[0084] It is to be understood that both the foregoing general description and the folowing 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 specificaly stated otherwise. As used herein, the use of “or” means “and / or” unless stated otherwise. Furthermore, the use of the term “including” as wel 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 specificaly stated otherwise.

[0085] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject mater described. Al 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. R3

[0086] In the various aspects described herein, R3can be hydrogen, halogen, -OR20, or -OR30.

[0087] In some embodiments, R3is -OR30, where R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R3is -OR30 and R30 is hydrogen or hydroxyl protecting group.

[0088] In some embodiments, R3is -OR30 and R30is a reactive phosphorus group. For example, R30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 P1 P2 2, -P(SR )N(R)2,-P(O)(OR )N(R)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT each RP1 is C1-6alkyl, optionaly 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; each RP2 is independently C1-6alkyl, optionaly 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, optionaly each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, preferably each RP2 is isopropyl; or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; and each RP3 is independently C1-30alkyl, C2-C30alkenyl, or C2-C30alkynyl, each optionaly independently 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— 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (CH2)p—NH2or CH2-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3, 4, 5 or 6, optionaly each RP3 is independently methyl, ethyl, propyl, isopropyl, n- butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0089] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P( P1 P2 P1 2 2 2 O)(SR)N(R)2, -P(O)(OR)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C alkyl, optionaly substituted with a CN or –SC(O)Ph; eac P2 1-6 h R is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0090] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SRP1)N(RP2 P1 2 2 2 )2, -P(O)(OR)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph; each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0091] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3 P2 P1 P2 P1 2 2 )N(R)2, -P(O)(SR)N(R)2, -P(O)(OR)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH P2 2CH2CN); each R is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0092] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N P2 P3 P2 P1 P2 P1 2 (R)2, -P(R)N(R)2, -P(O)(SR)N(R)2, -P(O)(OR)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently methyl, ethyl, propyl, isopropyl, n- butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0093] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P P3 P2 P1 P2 P1 2 2 (R)N(R)2, -P(O)(SR)N(R)2, -P(O)(OR)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CHCHCN); each RP2 is indepe P3 2 2 ndently isopropyl; and each R is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0094] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2)2, where: RP1 is 2- cyanoethyl (-CHCHCN); eac P2 2 2 h R is independently isopropyl.

[0095] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C alkyl, optio P2 1-6 naly substituted with a CN or –SC(O)Ph; both R taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0096] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P( P1 P2 P1 2 2 2 O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0097] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)( P1 P2 P1 2 2 2 SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0098] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0099] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SRP1)N(P2 P1 2 2 2 R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted4-8 membered heterocyclyl; other RP2 is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0100] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SP1 P2 P1 2 2 2 R )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0101] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2), - P1 P2 2 P(SR)N(R )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently optionaly substituted C1-6alkyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1- C6alkoxy.

[0102] In some embodiments, R3is -OR30 and R30 is -P(ORP1)N(RP2), -P(SRP1) P2 2 N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SRP1)N(RP2), -P(O)( P1 2 2 2 2 OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0103] In some embodiments, R3is -OR30 and R30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide. When R30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide, the internucleotide linkage between compound of Formula I and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage.

[0104] In some embodiments, R3is -OR30 and R30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage. For example, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide. In some embodiments, R30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage. In some other embodiments, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0105] In some embodiments, R3’is -OR30 and R30 is a hydroxyl protecting group. For example, R3’is -OR30 and R30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, 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, α-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-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, 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, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl alyl 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-1-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, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate.

[0106] In some embodiments, R3’is -OR30 and R30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionaly, R30 is TBDMS.

[0107] In some embodiments, R3is hydrogen or halogen. For example, R3 is H or F.

[0108] In some embodiments, R3is -OR20, where R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, such as C1-6alkoxyC1-6alkyl (e.g., 2-methoxyethyl) or N- (C alkyl)amin 3 1-6 ocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl). For example, Ris - OR20, and R20is hydrogen or hydroxyl protecting group.

[0109] In some embodiments, R3is -OR20, and R20is C1-6alkyl, optionaly substituted with 1, 2, 3, 4 or 5 substituents independently selected from OH, CN, SC(O)Ph, oxo (=O), SH, SO2NH2, SO2(C1-4)alkyl, SO2NH(C1-4)alkyl, halogen, carbonyl, thiol, cyano, NH2, NH(C1-C4)alkyl, N[(C1- )alkyl], C(O)NH, C(O)NH(C alkyl), C(O)N(C alkyl), CO 1-6 4 2 2 1-6 1-6 2 OH, COO(C alkyl) (e.g., COOMe), C2-6acyl (e.g., acetyl), (C1-8)alkyl, O(C1-8)alkyl (i.e., C1-8alkoxy), O(C1-8)haloalkyl, (C2-8)alkenyl, (C2-8)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 CH-aryl-alkoxy, where “m” and “p” are independently 1, 2, 3 2 3, 4, 5 or 6. For example, Ris - OR20, and R20is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionaly, R20 is methyl.

[0110] In some embodiments, R3is -OR20, and R20is C1-6alkoxyC1-6alkyl. For example, R3is -OR20, and R20is 2-methoxyethyl.

[0111] In some embodiments, R3’is -OR20 and R20 is a hydroxyl protecting group. For example, R3’is -OR20 and R20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl, p-nitrobenzyl, p- halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl, p-phenylbenzyl, 2-picolyl, 4- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT picolyl, 3- methyl-2-picolyl N-oxido, diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl, triphenylmethyl, α-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-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, 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, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl alyl 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-1-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, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate.

[0112] In some embodiments, R3’is -OR20 and R20 is TBDMS (or TBS), TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionaly, R20 is TBDMS (or TBS). R2

[0113] In the various aspects described herein, R2can be hydrogen, halogen, -OR20, or -OR30.

[0114] In some embodiments, R2is hydrogen or halogen. For example, R2is H or F.

[0115] In some embodiments, R2is -OR20, where R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, such as C1-6alkoxyC1-6alkyl (e.g., 2-methoxyethyl) or N- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (C1-6alkyl)aminocarbonylC1-6alkyl (e.g., 2-(N-methylamino)-2-oxoethyl). For example, R2is - OR20, and R20is hydrogen or hydroxyl protecting group. In other examples, R2is -OR20, where R20 is an optionaly substituted C1-6alkyl.

[0116] In some embodiments, R2is -OR20, and R20is C1-6alkyl, optionaly 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, R2is -OR20, and R20is methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, optionaly, R20 is methyl.

[0117] In some embodiments, R2is -OR20, and R20is C1-6alkoxyC1-6alkyl, optionaly 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, R2is -OR20, and R20is 2-methoxyethyl.

[0118] In some embodiments, R2’is -OR20 and R20 is a hydroxyl protecting group. For example, R2’is -OR20 and R20 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, 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, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT triphenylmethyl, α-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-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, 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, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl alyl 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-1-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, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate.

[0119] In some embodiments, R2’is -OR20 and R20 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionaly, R20 is TBDMS.

[0120] In some embodiments, R2is -OR30, where R30 is hydrogen, a hydroxyl protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide. For example, R2is -OR30 and R30 is hydrogen or hydroxyl protecting group.

[0121] In some embodiments, R2is -OR30 and R30is a reactive phosphorus group. For example, R30 is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2), - P1 P2 P1 P2 2 P(SR )N(R)2,-P(O)(OR )N(R)2, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, - P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly 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; each RP2 is independently C1-6alkyl, optionaly 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, optionaly each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, preferably each RP2 is isopropyl; or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; and each RP3 is independently C1-30alkyl, C2-C30alkenyl, or C2-C30alkynyl (e.g.,C1-10alkyl, C2- C10alkenyl, or C2-C10alkynyl , each optionaly independently 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, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 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, optionaly each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0122] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2), P1 P2 2 -P(SR )N(R)2, -P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph; each RP2 is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0123] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2), -P(SRP1)N(RP2 2 )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph; each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0124] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH P2 2CH2CN); each R is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0125] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2) P1 P2 P1 2 2 2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); each RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0126] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2,- P(O)(ORP1)N(RP2) P1 P2 P3 P2 P1 P2 P1 2, -P(S)(OR )N(R )2, -P(R )N(R )2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH P2 P3 2CH2CN); each R is independently isopropyl; and each R is independently 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0127] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2)2, where: RP1 is 2- cyanoethyl (-CH P2 2CH2CN); each R is independently isopropyl.

[0128] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1) P2 P3 P2 P1 P2 P1 2 N(R )2, -P(R )N(R )2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph; both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0129] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is C alkyl, optionaly substituted with P2 1-6 a CN or –SC(O)Ph; both R taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0130] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2)2, -P(S)(ORP1)N(RP2)2, -P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0131] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), -P(O)(SRP1)N(RP2 P1 2 2 2 )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: each RP1 is 2-cyanoethyl (-CH2CH2CN); both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0132] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2)2, -P(SRP1)N(RP2)2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2), - P1 P2 P1 2 2 2 P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 4-8 membered heterocyclyl; other RP2 is independently optionaly substituted C1-6alkyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0133] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N P2 P1 P2 P1 2 2 (R )2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently optionaly substituted C1-6alkyl.

[0134] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2) P1 P2 P1 2 2 2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently optionaly substituted C1-6alkyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1- C6alkoxy.

[0135] In some embodiments, R2is -OR30 and R30 is -P(ORP1)N(RP2) P1 P2 2, -P(SR)N(R )2,- P(O)(ORP1)N(RP2), -P(S)(ORP1)N(RP2), -P(RP3)N(RP2 P1 P2 P1 2 2 )2, -P(O)(SR )N(R )2, -P(O)(OR )H, - P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, where: RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl; other RP2 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl; and each RP3 is independently methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halogen, SH, or C1-C6alkoxy.

[0136] In some embodiments, R2is -OR30 and R30 is a bond to a nucleoside or a nucleotide, or an oligonucleotide. When R30 is a bond to a nucleoside, a nucleotide, or an oligonucleotide, the internucleotide linkage between compound of formulae I-II and the nucleoside, nucleotide, or oligonucleotide can be an unmodified (e.g., phosphodiester) internucleotide linkage or a modified (e.g., phosphorothioate) internucleotide linkage.

[0137] In some embodiments, R2is -OR30 and R30 is linked to 5’-position of a nucleoside, nucleotide, or oligonucleotide by an unmodified (e.g., phosphodiester) internucleotide linkage (e.g., or a modified (e.g., phosphorothioate) internucleotide linkage. For example, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide. In some embodiments, R30 is linked to the 5’- terminal (e.g., 5’-OH) of the oligonucleotide by an unmodified (e.g., phosphodiester) 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT internucleotide linkage. In some other embodiments, R30 is linked to the 5’-terminal (e.g., 5’-OH) of the oligonucleotide by a modified (e.g., phosphorothioate) internucleotide linkage.

[0138] In some embodiments, R2’is -OR30 and R30 is a hydroxyl protecting group. For example, R2’is -OR30 and R30 is a hydroxyl protecting group selected from the group consisting of BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t- butoxymethyl, POM, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2- chloroethoxy)methyl, SEMOR, THP, 3-bromotetrahydropyranyl, tetrahydrothiopyranyl, 1- methoxycyclohexyl, MTHP, 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl- S,S-dioxide, CTMP, 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a- octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p- methoxyphenyl, 2,4-dinitrophenyl, benzyl, 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, α-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-1- yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9- (9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl- S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, 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, Fmoc, alkyl ethyl carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl alyl 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-1-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, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT chlorodiphenylacetate, isobutyrate, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate.

[0139] In some embodiments, R2’is -OR30 and R30 is TBDMS, TBDPS, TMS, TES, TIPS, IPDMS, DEIPS, TBMPS, DPMS, dimethylthexylsilyl, tribenzylsilyl, tri-p-xylylsilyl, or triphenylsilyl, optionaly, R30 is TBDMS. RV

[0140] In some embodiments, at least one RV is independently a hydroxyl protecting group. For example, at least one RV, e.g., both RV are independently a hydroxyl protecting group selected from the group consisting of ethyl, pivaloyloxymethyl (POM), BOC or Boc, MOM, MTM, t-butylthiomethyl, SMOM, BOM, PMBM, p-AOM, GUM, t-butoxymethyl, siloxymethyl, MEM, 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, SEMOR, THP, 3- bromotetrahydropyranyl, tetrahydrothiopyranyl, 1-methoxycyclohexyl, MTHP, 4- methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl-S,S-dioxide, CTMP, 1,4-dioxan- 2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7- methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1- methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2- trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p-methoxyphenyl, 2,4- dinitrophenyl, benzyl, 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, α- 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-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4- methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl-S,S-dioxido, TMS, TES, TIPS, IPDMS, DEIPS, dimethylthexylsilyl, TBDMS, TBDPS, tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl, DPMS, 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, Fmoc, alkyl ethyl 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT carbonate, Troc, TMSEC, Psec, Peoc, alkyl isobutyl carbonate, alkyl vinyl carbonate alkyl alyl 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-1-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, (E)-2-methyl-2-butenoate, o-(methoxyacyl)benzoate, α- naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate,dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate, preferably the protecting group is POM or ethyl. Thus, in some embodiments, at least one RV, e.g., both RV are independently pivaloyloxymethyl (POM). In some other embodiments, at least one RV, e.g., both RV are independently.

[0141] In some embodiments, at least one RV, e.g., both RV are independently hydrogen. B (nucleobase)

[0142] In some embodiments of the various aspects described herein, B is an optionaly modified nucleobase. It is noted that the nucleobase can be a natural or non-natural nucleobase. By a “non-natural nucleobase” means 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 alyl 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- azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5- propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5- alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6- diaminopurine, 5-amino-alyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5- nitroindole, 3-nitropyrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5- methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5- methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 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 al which is incorporated herein by reference.

[0143] 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-(aminoalkyl)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- (alylamino)uracil, 5-(aminoalyl)uracil, 5-(aminoalkyl)uracil, 5-(guanidiniumalkyl)uracil, 5-(1,3- diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil, 5-(dialkylaminoalkyl)uracil, 5-(dimethylaminoalkyl)uracil, 5-(halo)uracil, 5-(methoxy)uracil, uracil-5-oxyacetic acid, 5-(methoxycarbonylmethyl)-2-(thio)uracil, 5-(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-(aminocarbonylethylenyl)- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 4-(thio)pseudouracil, 1-(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1-(aminoalkylamino-carbonylethylenyl)- 2(thio)-pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-4-(thio)pseudouracil, 1-(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1,3-(diaza)-2-(oxo)-phenoxazin- 1-yl, 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted 1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted 1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7- substituted 1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-(aminoalkylhydroxyl)-1,3-(diaza)-2- (oxo)-phenoxazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7- (aminoalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(aminoalkylhydroxyl)-1-(aza)-2- (thio)-3-(aza)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxyl)-1,3-(diaza)-2-(oxo)-phenoxazin-1- yl, 7-(guanidiniumalkylhydroxyl)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-(guanidiniumalkyl- hydroxyl)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-(guanidiniumalkylhydroxyl)-1-(aza)-2-(thio)- 3-(aza)-phenthiazin-1-yl, 1,3,5-(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, pyrolopyrizinyl, 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, O6-substituted purines, substituted 1,2,4-triazoles, and any O-alkylated or N-alkylated derivatives thereof.

[0144] 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.

[0145] 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 al of adenine, cytosine, guanine and uracil 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT without substantialy affecting the melting behavior, recognition by intracelular 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, pyrolopyrizinyl, 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.

[0146] In some embodiments, the natural or non-natural nucleobase is a protected nucleobase. As used herein, a “protected nucleobase” refers to a nucleobase comprising a nitrogen protecting group, and / or an oxygen protecting group, and / or a sulfur protecting group.

[0147] For example, the nucleobase is a pyrimidine modified at the C4 position. In another non-limiting example, the nucleobase is a pyrimidine modified at the C5 position.

[0148] In some embodiments, the nucleobase is a purine modified at the N2 position. In some embodiments, the nucleobase is a purine modified at the N6 position. For example, the nucleobase is a purine modified at the C6 position. In some non-limiting examples, the nucleobase is a N-7 deaza purine, optionaly modified at the N7 position.

[0149] In some embodiments, the nucleobase is a modified, protected or substituted analogs of a nucleobase selected from adenine, cytosine, guanine, thymine, and uracil. For example, the nucleobase is uracil, adenine, guanine, or cytosine, optionaly each independently comprising a hydroxyl, or amine protecting group. Double-stranded RNA

[0150] The skiled person is wel aware that double-stranded RNAs comprising a duplex structure of between 19 and 24, but specificaly 21, base pairs have been hailed as particularly efective in inducing RNA interference (RNAi). However, others have found that shorter or longer double-stranded oligonucleotides can be efective as wel. Accordingly, in some embodiments, a longer double-stranded oligonucleotide described herein is capable of inducing RNA interference. Stated another way, the longer double-stranded oligonucleotides described herein can mediate RNA interference. As used herein, the phrase “mediates RNAi” refers to the ability to inhibit or reduce the expression of a target nucleic acid, e.g., a target RNA such as a mRNA in a sequence specific manner.

[0167] Accordingly, in another aspect provided herein is a double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand substantialy or 100% (e.g., exactly) 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT complementary to the sense strand, and wherein one of the sense and antisense strand has a 5’- terminal modification comprising the structure:, wherein: X is O or S; and each RV is independently hydrogen or a hydroxyl protecting group.

[0151] In some embodiments, the 5’-terminal modification of the sense or antisense strand (e.g., the antisense strand) comprises the structure:

[0152] In certain embodiments, X is O. In certain embodiments, X is O and each RVis hydrogen. In certain embodiments, X is O and each RVis ethyl. In certain embodiments, X is O and each RVis pivaloyloxymethyl. X

[0153] It is noted that the sugar moiety of the nucleotide comprising themodification, i.e., the nucleotide at 5’-end of the sense or antisense strand (5’-terminal nucleotide) can comprise a 5- or 6- membered ring. For example, the sugar moiety of the nucleotide comprising the above modification can be a furanose (e.g., ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, including alpha and beta, D and L, deoxy, and modified derivates thereof), or a pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, alopyranose, altropyranose, gulopyranose, idopyranose, and talopyranose, including alpha and beta, D and L, deoxy, and modified derivates thereof).

[0154] Generaly, themodification replaces a CH2OH group on the sugar moiety of a 5’-terrminal nucleotide of the sense or antisense strand. For example, the4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTmodification replaces the 4’-CH2OH group on the furanose ring (e.g. ribofuranose, arabinofuranose, lyxofuranose, xylofuranose, ribulofuranose or xylulofuranose, preferably ribofuranose) or the 5’-CH2OH group on the pyranose (e.g., glucopyranose, galactopyranose, mannopyranose, alopyranose, altropyranose, gulopyranose, idopyranose, or talopyranose, preferably glucopyranose, galactopyranose, or mannopyranose) of the 5’-terminal nucleotide of the sense or antisense strand.

[0155] In some embodiments, the sense or antisense strand (e.g., the antisense strand) comprises at its 5’-end a compound of Formula (I) described herein. For example, the 5’-terminal nucleotide of the sense or antisense strand is of the structure:, wherein: each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); B is an optionaly modified nucleobase (e.g., uracil); one of R2and R3is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2- oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2- 6alkynyl (e.g., propargyl); and the other of R2and R3is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0156] In some embodiments, the 5’-terminal nucleotide of the sense or antisense strand(e.g., the antisense strand) has the 2 3one of Rand Ris -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0157] In some other embodiments, the 5’-terminal nucleotide of the sense or antisense strand (e.g., the antisense strand) has the 2 3one of Rand R is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0158] In some other embodiments, the 5’-terminal nucleotide of the sense or antisense strand (e.g., the antisense strand) has the structure: 2 3one of Rand R is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0159] In some other embodiments, the 5’-terminal nucleotide of the sense or antisense strand (e.g., the antisense strand) has the structure: 2 3one of Rand R is -OR30 (e.g., R3 is -OR30), wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

[0160] Preferably, the antisense strand of the dsRNA is an oligonucleotide described herein.

[0161] As used herein, the term “antisense strand” refers to an oligonucleotide that is substantialy or 100% (e.g., exactly) complementary to a target nucleic acid of interest. For example, an antisense strand can be complementary, in whole or in part, to target nucleic acid of interest, such as a messenger RNA, an RNA sequence that is not mRNA (e.g., microRNA, piwiRNA, tRNA, rRNA and hnRNA) or a sequence of DNA that is either coding or non-coding.

[0162] It is noted that each strand of the dsRNA can range from 12-40 nucleotides in length. For example, each strand independently can be between 14-40 nucleotides in length, 17-37 nucleotides in length, 25-37 nucleotides in length, 27-35 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, 21-23 nucleotides in length, 25-35 nucleotides in length, 26-35 nucleotides in length, 27-34 nucleotides in length, 28- 32 nucleotides in length or 29-31 nucleotides in length. Without limitations, the sense and antisense 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT strands can be equal length or unequal length. In some embodiments, the antisense strand is longer, e.g., by 1, 2, 3, 4, or 5 nucleotides than the sense strand.

[0163] In some embodiments, each of the sense and antisense strand is independently 15, 16, 17, 28, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. For, example, each of the sense and antisense strand is independently 18, 19, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, each strand is independently 19, 20, 21, 22 or 23 nucleotides in length. In some embodiments, one strand (e.g., the sense strand) is 18, 19, 20, 21 or 22 nucleotides in length and the other strand (e.g., the antisense strand) is 21, 22, 23, 24 or 25 nucleotides in length.

[0164] The sense and antisense strands of the dsRNA molecule are complementary to each other and can hybridize to each other to form a double-stranded or duplex region. Accordingly, the dsRNA molecule has a double-stranded or duplex region. The duplex region (double-stranded region) can be 17-25 nucleotide base pairs in length. For example, the dsRNA can have a duplex region of 17-24 nucleotide pairs in length. In some embodiments, the dsRNA has a duplex region of 18, 19, 20, 21, 22, 22, 23, 24, or 25 nucleotide base pairs in length. In some embodiments, the dsRNA has a duplex region of 19, 20, 21 or 22 nucleotide base pairs in length.

[0165] The dsRNA molecule can have one or more overhang regions (i.e., single-stranded region) and / or capping groups of dsRNA molecule at the 3’-end, or 5’-end or both ends of a strand. Without limitations, the overhang can be 1-3 nucleotides, e.g., 1, 2 or 3 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 sequence being targeted or it can be complementary to the sequence being targeted or can be other sequence. The sense and antisense strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers. Without limitations the overhang can be present at the 3’-end of only one of the strands or both strands.

[0166] In some embodiments, the dsRNA molecule comprises a single overhang. For example, the dsRNA molecule has a single overhang and the overhang is no more than one, two or three nucleotides in length. Preferably, the overhang is 2 nucleotides in length. In some embodiments, the overhang is present at the 3’-end of a strand (e.g., the antisense strand). In some embodiments, the dsRNA comprises a two-nucleotide overhang at the 3’-end of a strand (e.g., the antisense strand). For example, the overhang is present at the 3’-end of the antisense strand. For example, the antisense comprises a 1 or 2 nucleotide overhang at its 3’-end.

[0167] The dsRNA can also have a blunt end. For example, one end of the dsRNA is a blunt end and the other end has an overhang. Without limitations, the blunt end can be located at the 5’- end of the antisense strand (or the 3’-end of the sense strand) or vice versa. Generaly, the antisense strand of the dsRNA has a nucleotide overhang at the 3’-end, and the 5’-end is blunt. While not 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT bound by theory, the asymmetric blunt end at the 5’-end of the antisense strand and 3’-end overhang of the antisense strand favor the guide strand loading into RISC process. 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.

[0168] In some other embodiments, the dsRNA molecule has two blunt ends, i.e., at both ends of the dsRNA. For example, the two strands of the dsRNA are of the same length.In some embodiments, the antisense strand is of length 18 to 25 nucleotides. In some embodiments, the antisense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length. In some particular embodiments, the antisense strand is 23 nucleotides in length.

[0169] Similar to the antisense strand, the sense strand can be, in some embodiments, 18-25 nucleotides in length. In some embodiments, the sense strand is 21-25, 19-25, 19-21 or 21-23 nucleotides in length. In some embodiments, the sense strand is 21 nucleotides in length.

[0170] In some embodiments, sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length. Nucleic acid modifications

[0171] The longer double-stranded and single-stranded oligonucleotides described herein can comprise one or more nucleic acid modifications. Exemplary nucleic acid modifications include, but are not limited to, nucleobase modifications, sugar modifications, inter-sugar linkage modifications, conjugates (e.g., ligands), and any combinations thereof. It is noted that a nucleic acid modification(s) can be present in any position of longer double-stranded and single-stranded oligonucleotides. A nucleic acid modification(s) can be present in only one strand or both strands of a dsRNA. In some embodiments, only the antisense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications. In some embodiments, only the sense strand comprises at least one, e.g., two, three, four, five or more nucleic acid modifications. In some embodiments, both strands independently comprise at least one, e.g., two, three, four, five or more nucleic acid modifications.

[0172] Embodiments of the various aspects described herein recite specific position(s) on a strand, counting from an end of a strand. When the strand is single stranded, e.g., a longer-stranded oligonucleotide, the counting of the position is from the first nucleotide at the specified end. When the strand is part of a double-stranded molecule, e.g., a longer double-stranded oligonucleotide, the counting of the position can be from the first nucleotide at the specified end of the strand, or the first base-paired nucleotide in the strand at the specified end. Preferably, counting of the position is from the first nucleotide at the specified end of the strand. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT Thermaly destabilizing modifications

[0173] In some embodiments of any one of the aspects described herein, the dsRNA comprises a thermaly destabilizing modification. By a “thermaly destabilizing modification” is meant modification that result in a dsRNA having a lower overal melting temperature (Tm), preferably a Tm with one, two, three or four degrees lower, than the Tm of the dsRNA without having such a modification. Exemplary thermaly destabilizing modifications are described herein below, and can include, but are not limited to, abasic modifications; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2’-deoxy (i.e., 2’-H) modification, acyclic nucleotide (e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA), threose nucleic acid (TNA), a nucleotide linked by through its 2’-position (i.e., by its 2’-OH group to 5’-position of the subsequent nucleotide (a 2’-5’ RNA modification)); a Hyp-spacer modification; modified internucleotide linkages that decrease the thermal stability of dsRNA duplexes; or nucleobases with impaired W-C H-bonding to complementary base on the opposite strand.

[0174] In some embodiments, the dsRNA comprises at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more independently selected thermaly destabilizing modifications. The thermaly destabilizing modification can be present at any position of the dsRNA. Further, the thermaly destabilizing modifications al can be present in one strand or both strands of the dsRNA. In some embodiments, only the antisense strand comprises at least one, e.g., two, three, four or more thermaly destabilizing modifications. In some embodiments, only the sense strand comprises at least one, e.g., two, three, four or more thermaly destabilizing modifications. In some embodiments, both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermaly destabilizing modifications.

[0175] The thermaly destabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the thermaly destabilizing modification can occur on every nucleotide on the sense strand and / or antisense strand; each thermaly destabilizing modification can occur in an alternating patern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermaly destabilizing modifications in an alternating patern. The alternating patern of the thermaly destabilizing modifications on the sense strand can be the same or diferent from the antisense strand, and the alternating patern of the thermaly destabilizing modifications on the sense strand can have a shift relative to the alternating patern of the thermaly destabilizing modifications on the antisense strand.

[0176] In some embodiments, thermaly destabilizing modification is located at position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand. In some embodiments, the thermaly destabilizing modification is located at position 2, 3, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 4, 5 or 9 from the 5’-end of the antisense strand. In some other embodiments, the thermaly destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the antisense strand. In some particular embodiments, the thermaly destabilizing modification is located at position 7 from the 5’-end of the antisense strand.

[0177] In some embodiments, only the antisense strand comprises a thermaly destabilizing modification. For example, only the antisense strand comprises a thermaly destabilizing modification and said thermaly destabilizing modification is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the antisense strand, preferably the thermaly destabilizing modification is located at position 5, 6, 7, or 8; more preferably the thermaly destabilizing modification is located at position 6, 7, or 8. In some embodiments, only the antisense strand comprises a thermaly destabilizing modification and the thermaly destabilizing modification is located at position 7 of the antisense strand, counting from the 5’-end of the antisense strand.

[0178] Similar to the antisense strand, a thermaly destabilizing modification can be located at one of position 2, 3, 4, 5, 6, 7, 8 or 9, or preferably at position 4, 5, 6, 7, or 8, counting from the 5’- end of the longer-ssNA. In some embodiments, the thermaly destabilizing modification is located at position 2, 3, 4, 5 or 9 from the 5’-end of the longer-ssNA. In some other embodiments, the thermaly destabilizing modification is located at position 6, 7 or 8 from the 5’-end of the longer- ssNA. In some particular embodiments, the thermaly destabilizing modification is located at position 7 from the 5’-end of the longer-ssNA. Thermaly stabilizing modifications

[0179] In some embodiments, dsRNA comprises a thermaly destabilizing modification. By a “thermaly stabilizing modification” is meant modification that result in a dsRNA having a higher overal melting temperature (Tm), preferably a Tm with one, two, three or four degrees higher, than the Tm of the dsRNA without having such a modification. Exemplary thermaly destabilizing modifications are described herein below, and can include, but are not limited to, 2’-fluoro nucleotides (2’-F modifications), bridged nucleic acid (BNA), e.g., locked nucleic acid (LNA), and cyclohexene nucleic acid (CeNA). In some preferred embodiments, the thermaly stabilizing modification is a 2’-fluoro nucleotide. Exemplary, thermaly stabilizing modification are described herein below. Additional exemplary abasic nucleotides, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications are described in detail in WO 2011 / 133876 and WO2019222479, contents of both of which are incorporated herein by reference in their entireties.

[0180] In some embodiments, dsRNA can comprise at least two, e.g., three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen or more the thermaly stabilizing 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (e.g., 2’-F) modifications. Without limitations, the thermaly stabilizing (e.g., 2’-F) modifications al can be present in one strand or both strands of a dsRNA. In some embodiments, the sense strand comprises at least one, e.g., two, three, four or more thermaly stabilizing (e.g., 2’-F) modifications. In some embodiments, the antisense strand comprises at least one, e.g., two, three, four or more thermaly stabilizing (e.g., 2’-F) modifications. In some embodiments, both the sense and the antisense strands comprise at least one, e.g., two, three, four or more thermaly stabilizing (e.g., 2’- F) modifications. The thermaly stabilizing (e.g., 2’-F) modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the thermaly stabilizing (e.g., 2’-F) modification can occur on every nucleotide on the sense strand and / or antisense strand; each thermaly stabilizing (e.g., 2’-F) modification can occur in an alternating patern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise thermaly stabilizing (e.g., 2’-F) modifications in an alternating patern. The alternating patern of the thermaly stabilizing (e.g., 2’-F) modifications on the sense strand can be the same or diferent from the antisense strand, and the alternating patern of the thermaly stabilizing (e.g., 2’-F) modifications on the sense strand can have a shift relative to the alternating patern of the thermaly stabilizing (e.g., 2’-F) modifications on the antisense strand.

[0181] In some embodiments, the sense strand of the dsRNA comprises at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermaly stabilizing (e.g., 2’-F) modifications. In some embodiments, the sense strand comprises two, three, four, or five thermaly stabilizing (e.g., 2’-F) modifications. For example, the sense strand comprises three or four thermaly stabilizing (e.g., 2’-F) modifications. Without limitations, a thermaly stabilizing (e.g., 2’-F) modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises at least three thermaly stabilizing (e.g., 2’-F) modifications. For example, the sense comprises thermaly stabilizing (e.g., 2’-F) modification at least at positions 7, 10 and 11, counting from the 5’-end of the sense strand. In some other embodiments, the sense strand comprises at least four thermaly stabilizing (e.g., 2’-F) modifications. For example, the sense comprises thermaly stabilizing (e.g., 2’-F) modification at least at positions 7, 9, 10 and 11, counting from the 5’-end of the sense strand.

[0182] In some embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) 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 thermaly stabilizing (e.g., 2’-F) 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 thermaly stabilizing (e.g., 2’-F) modification. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0183] In some embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11 from the 5’-end, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In yet some other embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand.

[0184] In some embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In yet some other embodiments, the sense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 7, 9, 10, and 11, counting from the 5’-end of the sense strand, and the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’- end of the antisense strand.

[0185] In some embodiments, the sense strand does not comprise a thermaly stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermaly destabilizing modification of the duplex in the antisense strand.

[0186] The antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more thermaly stabilizing (e.g., 2’-F) modifications. In some embodiments, the antisense strand comprises two, three, four, five or six thermaly stabilizing (e.g., 2’-F) modifications. Without limitations, a thermaly stabilizing (e.g., 2’-F) modification in the antisense strand can be present at any position. In some embodiments, the antisense strand comprises at least three thermaly stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 14 and 16, counting from the 5’-end of the antisense strand. In some other embodiments, the antisense 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT comprises at least four thermaly stabilizing (e.g., 2’-F) modifications. For example, the antisense comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 14 and 16, counting from the 5’-end of the antisense strand. In some further embodiments, the antisense strand comprises at least five thermaly stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 9, 14 and 16, counting from the 5’-end of the antisense strand. In stil some further embodiments, the antisense strand comprises at least six thermaly stabilizing (e.g., 2’-F) modifications. For example, the antisense strand comprises thermaly stabilizing (e.g., 2’-F) modifications at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the antisense strand.

[0187] In some embodiments, the antisense strand comprises at least one thermaly stabilizing (e.g., 2’-F) modification adjacent to a stabilizing destabilizing modification. For example, the thermaly stabilizing (e.g., 2’-F) modification can be the nucleotide at the 5’-end or the 3’-end of the thermaly destabilizing modification, i.e., at position -1 or +1 from the position of the thermaly destabilizing modification. In some embodiments, the antisense strand comprises a thermaly stabilizing (e.g., 2’-F) modification at each of the 5’-end and the 3’-end of the thermaly destabilizing modification, i.e., positions -1 and +1 from the position of the destabilizing modification.

[0188] 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.

[0189] In some embodiments, the sense strand does not comprise a thermaly stabilizing (e.g., 2’-F) modification in position opposite or complimentary to the thermaly destabilizing modification of the duplex in the antisense strand. 2’-OMe nucleotides

[0166] In some embodiments, the dsRNA described herein can comprise at least one, e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more 2’-OMe nucleotides. Without limitations, the 2’- OMe nucleotides al can be present in one strand or both strands of a dsRNA. In some embodiments, both the sense and the antisense strands comprise at least one 2’-OMe nucleotide. The 2’-OMe modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2’-OMe modification can occur on every nucleotide on the sense strand and / or antisense strand; each 2’-OMe modification can occur in an alternating patern on the sense strand or antisense strand; or the sense strand and antisense strand both comprise 2’-OMe modifications in an alternating patern. The alternating patern of the 2’-OMe modifications on the sense strand 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT can be the same or diferent from the antisense strand, and the alternating patern of the 2’-OMe modifications on the sense strand can have a shift relative to the alternating patern of the 2’-OMe modifications on the antisense strand.

[0167] The antisense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen or more 2’-OMe modifications. Without limitations, a 2’-OMe modification in the antisense strand can be present at any position. In some embodiments, each nucleotide, except for any other specified modification (e.g., thermaly destabilizing modification(s), thermaly stabilizing modification(s), and / or 2’-deoxy (2’-H) modification(s)) of the antisense strand is independently a 2’-O-methyl nucleotide.

[0168] Like the antisense strand, the sense strand of the dsRNA molecule can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen or more 2’-OMe modifications. Without limitations, a 2’-OMe modification in the sense strand can be present at any positions. In some embodiments, each nucleotide, except for any other specified modification (e.g., thermaly stabilizing modification(s), lipophilic modification(s), inverted nucleotide(s), thermaly destabilizing modification(s), and / or 2’-deoxy (2’-H) modification(s) of the sense strand is independently a 2’-O-methyl nucleotide. 2’-deoxy (2’-H) nucleotides

[0169] In some embodiments, the dsRNA described herein can comprise a 2’-deoxy, i.e., 2’-H nucleotides. For example, the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) 2’-deoxy nucleotides.

[0170] A 2’-deoxy nucleotide can be present in any position of the sense or antisense strand. Further, 2’-deoxy nucleotides al can be present in one strand or both strands of the dsRNA.

[0171] In some embodiments, sense strand comprises 1, 2, 3, 4, 5 or more 2’-deoxy nucleotides. For example, the sense strand comprises a 2’-deoxy nucleotide at any one of positions 7, 9 and 11, counting from the 5’-end of the sense strand. In some embodiments, the sense strand comprises a 2’-deoxy nucleotide at least at position 9, counting from the 5’-end of the strand. For example, the sense strand comprises a 2’-deoxy nucleotide at least at positions 7 and 9, counting from the 5’-end of the strand. In another non-limiting example, the sense strand comprises a 2’- deoxy nucleotide at least at positions 9 and 11, counting from the 5’-end of the strand.

[0172] In some embodiments, antisense strand comprises 1, 2, 3, 4, 5, 6, 7, 8 or more 2’-deoxy nucleotides. For example, the antisense strand comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the antisense strand. In some embodiments, the antisense strand comprises a 2’-deoxy nucleotide at least at position 5, counting 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT from the 5’-end of the strand. For example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, counting from the 5’-end of the strand. In another non-limiting example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, and 12, counting from the 5’-end of the strand. In another non-limiting example, the antisense strand comprises a 2’-deoxy nucleotide at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’- end of the strand. Lipophilic modifications

[0173] In some embodiments, the dsRNA described herein can comprise a lipophilic modification. For example, the longer double-stranded and single-stranded oligonucleotides described herein can comprise at least one (e.g., 1, 2, 3, 4, 5 or more) lipophilic modifications. Exemplary lipophilic modifications include nucleotides modified with a lipophilic group, e.g., nucleotides comprising a lipophilic group (e.g., an C10-C30 alkyl, or a C10-C30 alkenyl group, such as a C16 alkyl, a C16 alkenyl, a C18 alkyl, a C18 alkenyl, a C20alkyl, a C20 alkenyl, a C22 alkyl, a C22 alkenyl, a C24 alkyl, a C24 alkenyl; C15 alkyl, a C15 alkenyl, a C17 alkyl, a C17 alkenyl, a C19alkyl, a C19 alkenyl, a C21 alkyl, a C21 alkenyl, a C23 alkyl, or a C23 alkenyl) at their 2’-position. Some exemplary lipophilic nucleotides include, but are not limited to, 2’-O-hexadecyl-modified nucleotide (Nhd), 2’-O-docosanyl-modified nucleotide (Nda), 2’-O-(omega-hydroxy-hexadecyl)- modified nucleotide (NhdOH), and 2’-O-(omega-hydroxy-docosanyl)-modified nucleotide (NdaOH).

[0174] A lipophilic modification can be present in any position of the sense or antisense strand. Further, lipophilic modifications al can be present in one strand or both strands of a dsRNA. In some embodiments, only the sense strand comprises a lipophilic modification. For example, the sense strand comprises a lipophilic modification at any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand. In some embodiments, the sense strand comprises a lipophilic modification at any one of positions 4, 5, 6, 7, 8, 13, 14, 15, 16, 17 or 18, counting from the 5’-end of the sense strand.

[0175] In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl, 2’-fluoro, 2’-deoxy, LNA, HNA, CeNA, 2’- methoxyethyl, 2’-O-alyl, or 2’-C-alyl, 2’-deoxy, or. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl or 2’-fluoro. It is to be understood that these modifications are in addition to any other specified modification (e.g., at least one thermaly destabilizing modification of the duplex present in the antisense strand) of dsRNA molecule. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0176] In some embodiments, at least two diferent modifications are typicaly present on the sense strand and antisense strand. Those two modifications may be the 2’-deoxy, 2’-O-methyl or 2’-fluoro modifications, thermaly destabilizing modifications. In some embodiments, the sense strand and antisense strand each comprises two diferently modified nucleotides selected from 2’- O-methyl or 2’-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, each residue of the sense strand and antisense strand is independently modified with 2’- O-methyl nucleotide, 2’-deoxy nucleotide or 2´-deoxy-2’-fluoro nucleotide. Again, it is to be understood that these modifications are in addition to any thermaly destabilizing modification of the duplex present in the antisense strand.

[0177] In some embodiments, the antisense strand comprises at least one thermaly destabilizing modification, and the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide, 2’-O-N-methylacetamido (2’-O- NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O- aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, the antisense strand comprises a thermaly destabilizing modification and the remaining nucleotides are independently a 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro nucleotide. In some embodiments, the antisense strand comprises: (i) a thermaly destabilizing modification at position 5, 6, 7, or 8, counting from the 5’-end of the antisense strand; (i) at least two, e.g., 3, 4, 5 or 62’- fluoro nucleotides; and (ii) the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide.

[0178] In some embodiments, each nucleotide of the sense strand is independently 2’-O- methyl nucleotide, 2’-deoxy nucleotide, 2´-deoxy-2’-fluoro (2’-F) nucleotide, 2’-O-N- methylacetamido (2’-O-NMA) nucleotide, a 2’-O-dimethylaminoethoxyethyl (2’-O-DMAEOE) nucleotide, 2’-O-aminopropyl (2’-O-AP) nucleotide, or 2’-ara-F nucleotide. For example, each nucleotide of the sense strand is independently 2’-O-methyl nucleotide, 2’-deoxy nucleotide, 2´- deoxy-2’-fluoro nucleotide. In some embodiments, the sense strand comprises at least two, e.g., 3, 4, 5 or 62’-fluoro nucleotides, and the remaining nucleotides are independently a 2’-O-methyl nucleotide or 2’-deoxy nucleotide.

[0179] In some embodiments, at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5’- end of the antisense strand of a dsRNA can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT the 5’-end of the duplex. In some embodiments, the nucleotide at the 1 position within the duplex region from the 5’-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5’- end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5’- end of the antisense strand is an A:U base pair. It is noted that either the sense strand or the antisense strand can comprise the adenosine (A) nucleotide. Modified internucleotide linkages

[0180] The dsRNA described herein can comprise at least one, e.g., two, three, four, five, six, seven, eight, nine, ten or more modified internucleoside linkages. As used herein, “internucleoside linkage” refers to a covalent linkage between adjacent nucleosides. Exemplary modified internucleoside linkage include, but are not limited to, phosphodietetrs, phosphorothioates (R, S, or racemic), phosphorodithioates, methylenemethylimino (MMI, 3'-CH2-N(CH3)-O-5'), phosphotriesters, alkylphosphonates (e.g., methylphosphonates), phosphoramidate, methylenemethylimino (—CH2-N(CH3)-O—CH2-), thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—), siloxane (—O—Si(H)2-O— and dialkylsiloxane), N,N′-dimethylhydrazine (—CH2-N(CH3)-N(CH3)-), amide-3 (3'-CH2-C(=O)-N(H)-5'), 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, 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', 3'- NHP(O)(OCH3)-O-5’), imidophosphoramidate (“imidp”), 2’->5’ internucleoside linkages, 2’->3 internucleoside linkages, 3’->3 internucleoside linkages, and 5’->5’ internucleoside linkages, optionaly the modified internucleoside linkage is phosphorothioate, methylphosphonate, imidp or MMI, more preferably the modified internucleoside linkage is phosphorothioate (PS).

[0181] A modified internucleotide linkage can occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification can occur on every nucleotide on the sense strand and / or antisense strand; each internucleotide linkage modification can occur in an alternating patern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating patern. The alternating patern of the internucleotide linkage modification on the sense strand can be the same or diferent from the antisense strand, and the 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT alternating patern of the internucleotide linkage modification on the sense strand can have a shift relative to the alternating patern of the internucleotide linkage modification on the antisense strand.

[0182] In some embodiments, the dsRNA comprises the modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage(s) in the overhang region. For example, the overhang region comprises two nucleotides having modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between the two nucleotides. Internucleotide linkage modifications can also be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or al the overhang nucleotides can be linked through modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage, and optionaly, there can be additional modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage 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 can be at the 3’-end of the antisense strand.

[0183] With respect to position of an internucleotide linkage, the indicated position refers to the internucleotide linkage that links the nucleotide at said position with the nucleotide one position down stream from said position. In other word, an internucleotide linkage at position N means it is between nucleotides N and N+1. Thus, an internucleotide linkage at position 1, counting from the 5’-end, means the linker is between the nucleotides at positions 1 and 2, counting from the 5’- end.

[0184] In some embodiments, the sense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the sense strand. For example, the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the sense strand, and sense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the sense strand. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0185] In some embodiments, the antisense strand comprises one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand, and one to five (e.g., 1, 2, 3, 4 or 5) modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkages within position 1-5, counting from the 5’-end of the antisense strand. For example, the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 5’-end of the antisense strand, and the antisense strand further comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, counting from the 3’-end of the antisense strand.

[0186] In some embodiments, the sense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the sense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the sense strand; and the antisense strand comprises a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2 counting from the 5’-end of the antisense strand, a modified internucleoside (e.g., phosphorothioate or methylphosphonate internucleotide) linkage at positions 1 and 2, counting from the 3’-end of the antisense strand. Overhang modifications

[0187] The nucleotides in the overhang region of the dsRNA molecule 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- methoxyethyladenosine, 2’-O-methoxyethyl-5-methylcytidine, GNA (glycol nucleic acid), SNA (serinol nucleic acid), TNA (threose nucleic acid), and any combinations thereof. For example, TT (or UU) can be an overhang sequence for either end on either strand. The 5’- or 3’- overhangs at the sense strand, antisense strand or both strands of the dsRNA molecule can be phosphorylated. In some embodiments, the overhang region contains two nucleotides having a phosphorothioate internucleotide linkage between the two nucleotides, where the two nucleotides in the overhang region can be the same or diferent. 5’-modifications 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT X

[0188] The 5’-end of a strand of the dsRNA lacking the(e.g.,modification can also be modified. Exemplary modifications for the 5’-end include, but are not limited a 5’-morpholino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a morpholino group), a 5’-dimethylamino nucleotide (e.g., a nucleotide where the 5’-OH group is replaced with a dimthylamino group, a 5’- deoxy nucleotide, an inverted nucleotide (i.e., a nucleotide linked via a 5’->5’ linkage to the rest of the strand), an inverted abasic nucleotide (e.g., an abasic nucleotide linked by a 5’->5’ linkage), or an inverted abasic locked nucleic acid modification (i.e., an LNA lacking a nucleobase and linked by a 5’->5’ linkage) at the 5’-end.

[0189] In some embodiments, the sense strand of the dsRNA comprises a 5’-morpholino nucleotide, a 5’-dimethylamino nucleotide, a 5’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end. For example, the sense strand comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end. For example, the sense strand of the dsRNA comprises an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 3’-end.

[0190] In some embodiments, the sense strand comprises a ligand at its 3’-end. Ligands

[0191] In some embodiments, the longer double-stranded and single-stranded oligonucleotide can comprise a ligand. Without wishing to be bound by a theory, a ligand can modify one or more properties of the atached molecule (e.g., the dsRNA described herein) including but not limited to pharmacodynamic, pharmacokinetic, binding, absorption, celular distribution, celular uptake (cel targeting), charge and clearance.

[0192] In some embodiments, the ligand is a targeting ligand. As used herein the term “targeting ligand” refers to any molecule that provides an enhanced afinity for a selected target, e.g., a cel, cel type, tissue, organ, region of the body, or a compartment, e.g., a celular, 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. Carbohydrate based targeting ligands include, but are not 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 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 andlectins. 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 scafold molecule.

[0193] In some embodiments, the ligand is an asialoglycoprotein receptor (ASGPR) ligand. By an ASGPR ligand is meant a ligand that binds the ASGPR. In some embodiments, the ASGPR ligand comprises one or more (e.g., 1, 2, 3 or more) GalNAc or GalNAc derivatives atached through a bivalent or trivalent branched linker. An exemplary ASGPR ligand is:4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT, or a loop forming oligonucleotide where 3 or 4 consecutive nucleosides are modified with GalNAc containing ligands; such as, for example, the loop containing oligonucleotide,each G* and A* is substituted at the 2’-O position with.

[0194] Exemplary carbohydrate based targeting ligands are also described in US Patent No. 5,994,517 and US Patent No.6,906,182, and PCT Application No. PCT / US2022 / 047102, contents of each of which are incorporated herein by reference in their entireties. Exemplary folate and 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT folate analogs targeting 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.

[0195] It is noted that when two or more ligands are present, the ligands can al have same properties, al have diferent properties or some ligands have the same properties while others have diferent properties. In a preferred embodiment, al the ligands have diferent properties.

[0196] It is noted that a ligand can be linked at any position of either strand of the dsRNA. For example, the ligand can be at the 5’-end, 3’-end or at an internal position of a strand, e.g., sense or antisense strand of the dsRNA.

[0197] In some embodiments, the sense strand comprises the ligand, i.e., the ligand is conjugated to the sense strand. For example, the ligand is conjugated to the 3′ end of the sense strand. Exemplary sense strands

[0198] In some embodiments, the sense strand of the dsRNA has one of the folowing modification paterns: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTf f f f f f f f f f f f f 6 n n n n n N sf n n N n N N n N N n n n n n n n N N N N d N N N Nito f f f f f f f f fm 5 n n N N n n n n n n n n n n N n N N n N N n n n n n n n N Ndn f f f f f f f a 4 n n n n n n n n n N N n n n n n n n n n n N n N N n N N n nrts f f f f fes 3 n n N N n n n n n n n n n n n n N N n n n n n n n n n n N nnes f f f f y 2 n n n n n n n n n N N n n n n n n n n n n n n N N n n n n nral f f f f p 1 n n N N n n n n n n n n n n n n N N n n n n n n n n n n n nme d x -E ’ o 3.: 5 m 87B 8 01 2345678901 234567890 3-el 1 234567891 1 1 1 1 1 1 1 1 1 222 87b S S S S S S S S S S S S S S S S S S S S S S2 S2 S2 S2 S2 S2 S2 S3 S 91-a 1 T # 684Aty. Dkt. No.051058-000104WOPT do m-’ 312 02 91 81 7 f f 1 N N n n n 61 n n n n n 5 f f 1 N N n n n 41 n n n n n 3 f f 1 N N n n n 21 n n n n n 1 f 1 N n n n n 01 n n n n n f f 9 N N n n n 8 n n n n n f f NNf 7 N N d d N f f f 6 N N n N n f f Nf f 5 N N d N N 4 n n n n n f f f f 3 N N n N N 2 n n n n n f f 1 N N n n n - d ’ o 5 m 3.878 1 234 3- 33335 87 S S S S3 S 91- # 1684Aty. Dkt. No.051058-000104WOPT wherein n is a 2’-O-methyl-nucleotide; (dN) is a 2’-deoxy-nucleotide; Nf is a 2’-fluoro-modified nucleotide (e.g., 2’-deoxy-2’-fluoro modified nucleotide); and the sense strand optionaly comprises either: (a) a 3’-terminal or 5’-terminal modification selected from: (i) 5’-(L1)- atached to the 5’-terminal nucleotide (e.g., through the 5’-O of the terminal nucleoside), optionaly via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; (i) -(L2)-3', atached to the 3’-terminal nucleotide (e.g., through the 3’-O of the terminal nucleoside), optionaly via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; (ii) 5’-(L1)(I)- atached to the 5’-terminal nucleotide (5’-5’), optionaly via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; or (iv) -(I)(L2)-3', atached to the 3’-terminal nucleotide (3’-3’), optionaly via a divalent linking group, such as a phosphodiester or phosphorothioate linkage; wherein each (I) is an inverted nucleotide (e.g., an inverted abasic nucleotide, such as an inverted abasic ribonucleotide, such as an inverted abasic deoxyribonucleotide); (L1) and (L2) are independently hydrogen or a group comprising a ligand (L), wherein the ligand is selected from: (i) a lipophilic group; examples include a group comprising an C10-C30 alkyl, or a C10-C30 alkenyl group, e.g., a C10 alkyl, C10 alkenyl, C12 alkyl, C12 alkenyl, C14 alkyl, C14 alkenyl, C15 alkyl, C15 alkenyl, C16 alkyl, a C16 alkenyl, a C18 alkyl, a C18 alkenyl, a C20alkyl, a C20 alkenyl, a C22 alkyl, a C22 alkenyl, a C24 alkyl, a C24 alkenyl; C15 alkyl, a C15 alkenyl, a C17 alkyl, a C17 alkenyl, a C19alkyl, a C19 alkenyl, a C21 alkyl, a C21 alkenyl, a C23 alkyl, or a C23 alkenyl group; examples include, but are not limited to, a hexadecyl group, a docosanyl group, an omega-hydroxy-hexadecyl group, and an omega-hydroxy-docosanyl group; or (i) a receptor targeting ligand, such as a group comprising an ASGPR ligand; or (ii) a precursor functional group, where the precursor functional group is suitable for post-synthetic functionalization with a ligand (e.g. as descirbed in (i) or (i)) 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT containing or conjugated to a complementary reactive functional group; examples of precursor functional groups include, but are not limited to, amino, carboxy, primary amido (-C(O)NH2), N-succinamido, azido (-N3), mercapto (- SH), active esters (e.g., an N-hydroxysuccinimde ester (NHS ester) or a pentafluorophenyl ester), 1,2,4,5-tetrazinyl (e.g., 3-methyl-1,2,4,5-tetrazinyl, 3-(pyridin-2-yl)-1,2,4,5-tetrazinyl, or 3-(pyrimidin-2-yl)-1,2,4,5-tetrazinyl-); cyclooctynyl (e.g., bicyclo[6.1.0]nonynyl (BCN) or dibenzocyclooctynyl (DBCO)), trans-cyclooctenyl, 2-methylsulfonylpyrimidinyl, 4-vinylpyridinyl, and protected forms thereof; or (b) a nucleotide comprising a ligand modification, as described above, that replaces the nucleotide decribed above; examples of lipophile modified nucleotides include, (Nhd) – 2’-O-hexadecyl-modified nucleotide; (Nda) – a 2’-O-docosanyl-modified nucleotide; (NhdOH) – 2’-O-(omega-hydroxy-hexadecyl)-modified nucleotide); or (NdaOH) – a 2’- O-(omega-hydroxy-docosanyl)-modified nucleotide.

[0199] In each of the preceding sense strands, each of the nucleotides are connected in series (i.e., in a 3’->5’ manner) via optionaly modified internucleotide linkages. For example, each of the nucleotides are connected by phosphodiester or phosphorothioate internucleotide linkages.

[0200] In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkage linkage s, counting from the 5’-end of the oligonucleotide.

[0201] In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester linkages, counting from the 5’-end of the oligonucleotide.

[0202] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide.

[0203] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide.

[0204] In other embodiments, each (inv) atached to a 5’-terminal nucleotide is connected via a phosphorothioate linkage (5’-5’).

[0205] In other embodiments, each (inv) atached to a 3’-terminal nucleotide is connected via a phosphorothioate linkage (3’-3’).

[0206] In certain other embodiments, each (inv) atached to a 5’-terminal nucleotide is connected via a phosphorothioate linkage (5’-5’), and each (inv) atached to a 3’-terminal nucleotide is connected via a phosphorothioate linkage (3’-3’).

[0207] in certain embodiments, the sense strand is according to any one of S1-S35 wherein the nucleotide at any one of positions 4-8 or 13-18 counting from the 5’-end of the strand, is replaced with a nucleotide that is substituted with an (L) group.

[0208] For example, in certain embodiments, the sense strand of the dsNA has one of the folowing modification paterns: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT do ) ) ) ) ) ) ) m2222222 -’ L 3 ( L ( L ( L ( L ( L ( L (( m dn 6 n n n n n n n n n n n n )L n n n n n n n ) a ( n n n n n L n n n r ( ts es 5 f f n n n n n n n n ) ) n n L n n n n n n n n n n n n L e N N ( ( n n n n s y 4 ) ) r n n n n n n n n n n L n n n n n n n n n n n n L n n n n n al ( ( p m 3 f f n n n n n n n )L n n n n n n n n n n n n )L n n n n n n e N N ( ( xel 2 s n s n s n s n s n s n s n s s n ) s n s n s s n s n s s s s s s s s ) s s s s s s s a L n n n n n n n n L n n n n n n n n ( ( oiti s s s 1 f f s n s s s s ) s s s s s s s s s s s s s) s s s s s s s s d N N n n n n L ( n n n n n n n n n n n n L n n n n n n n n d ( A 3. : d 87 C o 83- e 8 l m b - 7 ’ 91- a 5 1 T 684 # 637383930414243444546474849405152535455565758595061626364 S S S S S S S S S S S S S S S S S S S S S S S S S S S S6 SAty. Dkt. No.051058-000104WOPT6 n n n n n n n n n n n n n n n n n n n n n n n n n n n n )L ( n n n n 5 n n n n n n n n n n n n n n n f f f f f f f f f f f f f f f f N N n N N n N N N N N N N N N N N N 4 n n n n n n n n n n n n n n n n n n n n n n n n n n n ) L ( n n n n n 3 n n n n n n n n n n n n n n n f f N N n n n n n n n n n )L ( n n n n n n 2 s s s s s n n n n n n n n n n n n n n n n ) n n n n n n n n n L ( n n n n n n n s n s f f ) 1 n s n s n s n n n n n n n n n n n N N n n n n n n n L ( n n n n n n n n d )s ) o ( s ) ) ( ) s ) ( s ) ) ) ) 3. ( s( s( s( s( 878 m I ) - ( I( s( )I ) ) ( I( s( )I ) ) ) 3 ( I I I -8 ’ )1 )1 )I ( )1 )1 )I ( ) ( 1 ) ( 1 ) ( 1 ) 7 1 91 5 L ( L ( L ( L ( L ( L ( L ( L - ( 1684 #566676869601 2345678901 2345678901 234567 S S S S S7 S7 S7 S7 S7 S7 S7 S7 S7 S7 S8 S8 S8 S8 S8 S8 S8 S8 S8 S8 S9 S9 S9 S9 S9 S9 S9 S9 SAty. Dkt. No.051058-000104WOPT1 n n n n n n n n n n n n n n n n n n n n n n n n n n 9 f f f f f f f f f f f f f f f f f f f f f f f N N N N N N N N N N N N N N N N N N N N N N NN d N d N d 8 f f f f f f f f f f f f f f f f f f f f f f N N N N N N N N N N N N N N N N N N N N N N n n n n 7 f f f f f f f f f f f f f f f f f f f f f f f NNf N N N N N N N N N N N N N N N N N N N N N N N d d N 6 n n n n n n n n n )L ( n n n n n n n n n n n n n n n n 5 f f n n n n n n )L n n f f f f f N N ( n n n n n n n n N N N n n N N 4 n n n n n n n ) L ( n n n n n n n n n n n n n n n n n n 3 n n n n n n )L ( n n n n n n n n n n n n n n n n n n n 2 n n n n n )L ( n n n n n n n n n n n n n n n n n n n n ) 1 n n n n L ( n n n n n n n n n n n n n n n n n n n n n 3. d 87 o 83- m 8 - 7 ’ 91 5 -1684 #890010203040506070809001 1 2345678901 23 991 1 1 1 1 1 1 1 1 1 1 11 11 11 11 11 11 1 1 1 2222 S S S S S S S S S S S S S S S S S S S1 S1 S1 S1 S1 S1 S1 SAty. Dkt. No.051058-000104WOPT wherein n is a 2’-O-methyl-nucleotide; (dN) is a 2’-deoxy-nucleotide; Nf is a 2’-fluoro-modified nucleotide; (L) is a ligand-modified nucleotide, e.g., comprising a lipophilic group (e.g., C16 or C22 modification) or an ASGPR ligand; and s is a phosphorothioate internucleotide linkage, (s) is a phosphorothioate or a phosphodiester internucleotide linkage (e.g., in certain embodiments, each (s) is a phosphorothioate); (I) is an an inverted abasic nucleotide (e.g., an inverted abasic ribonucleotide or an inverted abasic deoxyribonucleotide); (L1) and (L2) are independently hydrogen or a group comprising a ligand, and sense strand optionaly comprises a 5’-morpholino nucleotide, a 5’-dimethylamino nucleotide, a 5’-deoxy nucleotide, an inverted nucleotide, an inverted abasic nucleotide, or an inverted abasic locked nucleic acid modification at the 5’-end.

[0209] In some embodiments, the antisense strand of the dsNA has one of the folowing modification paterns: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTN N N N N N N N N N N N N N Nsfit 7 N d N d n n n n n n n n n n n N d N d n n n n n n n n n n n N d N d n n no m d 6 n n f f f f f f f f f n n n n f f f f f f f f f n n n n f f f N N N N N N N N N N N N N N N N N N N N N narts 5 N d N d n n n n n n n n n n n N d N d n n n n n n n n n n n N d N d n n nesn 4 n n n n n f f f f f f f f f f f f f f f f N N N N n n n n n n n n n ne N N N N N N N N N N N N sitn 3 n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n na y 2 NN f f f f f f f f f f f f f f f f f f f f N N N N NN f f NNf f fr N N N N N N N N N N N N N N N N N a d d d d N d d N N Nlp 1 n n n n n n n n n n n n n n n n n n n n n n n n n n n n n n nmex -’ d E o Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z 3.: 5 m 87D 83el 01 2345678901 2345678901 -87b # 1S2S 3S4S5S 6S7S8S 9S1S1S1S 1S1S1S 1S1S1S1S 2S2S2S 2S2S2S 2S2S2S2S 3S3S 91-a 1 T A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 684Aty. Dkt. No.051058-000104WOPT do m-’ 3 3222 12 02 91 n n n n n n n n 8 f f f f f f f 1 N N N N N N N n 71 n n n n n n n n 6 f f f f f f f f 1 N N N N N N N N 51 n n n n n n n n 4 f f f f f f f f 1 N N N N N N N N 31 n n n n n n n n 2 f f f f 1 N N n n n n N N 11 n n n n n n n n 0 f f f f 1 N N N N n n n n 9 n n n n n n n n 8 f f f f f f N N N N N N n n 7 n n n n n n n n 6 f f f f f f N N N N N N n n 5 n n n n n n n n 4 f f f f f f f f N N N N N N N N 3 n n n n n n n n 2 f f f f f f f f N N N N N N N N 1 n n n n n n n n -’ d 5 o Z Z Z Z Z Z Z Z 3 m .8783 23456789 -8 # 3S3S3S3S 3S3S3S 3 7 S 91- A A A A A A A A 1684Aty. Dkt. No.051058-000104WOPT wherein: n is a 2’-O-methyl-modified nucleotide; s is a phosphorothioate internucleotide linkage (3’- 5’); (dN) is a 2’-deoxy-nucleotide; Nf is a 2’-fluoro-modified nucleotide;X is O or S; and each RV is independently hydrogen or a hydroxyl protecting group; and where the preceding structures replaces the 4’-CH2OH group on the furanose (e.g., ribose) or 5’-CH2OH group on the pyranose ring of the 5’-terminal nucleotide.

[0210] In each of the preceding antisense strands, each of the nucleotides are connected in series (i.e., in a 3’->5’ manner) via phosphodiester or phosphorothioate internucleotide linkages.

[0211] In one embodiment of any one of AS1-AS39, the nucleotide at one of positions 5-8, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G), such as: (Ngn) – a glycol nucleic acid, S-isomer; (N2p) – a 2′-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively); (Tna) – a threose nucleotide (connected by 3’-3’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively); (MM) a nucleobase mismatch to the sense strand; or (Nul) an unlocked nucleic acid.

[0212] In one embodiment of any one of AS1-AS39, the nucleotide position 5, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS1-AS39, the nucleotide position 6, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS1-AS39, the nucleotide position 7, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS1-AS39, the nucleotide position 8, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0213] For example, the antisense strand may be selected from any one of AS40 - AS58 as recited in Table E: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT -’ 3 32 22 12 02 91 81 71 6151 n n n n n n n n n n n n n n n n n n 4 f f f f f f f f f f f f f f f f f f 1 N N N N N N N N N N N N N N N N N N 31 n n n n n n n n n n n n n n n n n n 2 N f N f f 1 d n n n n N d n n n n NN d n n n n N 1 1 n n n n n n n n n n n n n n n n n n 01 n n n n n n n n n n n n n n n n n n 9 n n n f f f f f f N N n n n n N N n n n n N N n 8 n n n n f n n n n n f n f N N n n n n N n 7 G G G G G G G G G G G G G G G G G G 6 n n f f f n n f f f f f f N N N n N N N n n n N N N n 5 N d N d n n n n N d N d n n n n N d N d n n n n 4 n n n n n f n n n n n f n n n n n f N N N 3 n n n n n n n n n n n n n n n n n n 2 N f f f f f f f f f f f d N d N NN f f f f N N N N N N N N d N N N N N 1 n n n n n n n n n n n n n n n n n n -’ d 5 o Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z 3.. m 87E 83el 01 2345678901 234567 -87b # 4S 4S4S4S4S 4S4S4S 4S4S5S 5S5S5S5S 5S5S5S 91-a T A A A A A A A A A A A A A A A A A A 1684Aty. Dkt. No.051058-000104WOPT

[0214] In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide. In certain embodiments, the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide.

[0215] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide.

[0216] In certain embodiments, where the nucleotide in m nucleotides in length, the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage, and the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide. That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 21 and 22 are connected by a phosphorothioate internucleotide linkage; and positions 22 and 23 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide; and for a nucleotide in 21 nucleotides in length, the nucleotides at positions 20 and 21 are connected by a phosphorothioate internucleotide linkage, counting from the 5’-end of the oligonucleotide.

[0217] In certain embodiments, where the nucleotide in m nucleotides in length: (a) the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; (b) the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; (c) the nucleotides at positions m-2 and m-1 are connected by a phosphorothioate internucleotide linkage; and 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (d) the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide.

[0218] That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 21 and 22; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide. And, for a nucleotide in 21 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 19 and 20; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide.

[0219] In certain embodiments, where the nucleotide in m nucleotides in length: (a) the nucleotides at position 1 and 2 are connected by a phosphorothioate internucleotide linkage; (b) the nucleotides at position 2 and 3 are connected by a phosphorothioate internucleotide linkage; (c) the nucleotides at position 3 and 4 are connected by a phosphorothioate internucleotide linkage; and (d) the nucleotides at positions m-1 and m are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide.

[0220] That is, for a nucleotide in 23 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 22 and 23 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide. And, for a nucleotide in 21 nucleotides in length, the nucleotides at positions 1 and 2; 2 and 3; 3 and 4; and 20 and 21 are connected by a phosphorothioate internucleotide linkage, and the remaining nucleotides are connected in via phosphodiester bonds, counting from the 5’-end of the oligonucleotide.

[0221] For example, in some embodiments, the antisense strand of the dsNA has one of the folowing modification paterns in Table F. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT -’ sfito m 6 n n f f f f f f f f f N N N N N N n n f f f f f f f f f f f N N N n n N N N N N N N N N n n n n N Nesne 5 NN n n n n n n n n n n n NN n n n n n n n n n n n NN n ns d d d d d d itn f f f f f f f f a 4 n n n n n n n f f f f f f f f N N N N N N N N n n n N N N N N N N N n n n nyral 3 n n n n n n n n n n n n n n n n n n n n n n n n n n n n n np m s s sf sf sf sf sf sf sf sf sf sf sf s s sf sf sf sf s s s s s s s s s s se 2 NN f f f f f f f f f N NN NN xe d d N N N N N N N N N N d d N N N N N N N N N N N d d N Nla 1 s n s n s n s n s n s n s s s s s s s s s s s s s s s s s s s s s s s sn n n n n n n n n n n n n n n n n n n n n n n n noitid -’ do d 5 Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z mA 3.: 87F 83el 8595061626364656667686960717273747576777879708182838485 - 86878 879b # SSSSSSSSSSSSSSSSSSSSSSSSSSSSSS 1-a T A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 1684Aty. Dkt. No.051058-000104WOPT -’ d 3 o m 32 22 12 02 91 81 71 61 51 41 31 21 11 01 9 8 76 f f f f f f f n n n n f f f f f f f f f n n f f f f f f f N N N N N N N N N N N N N N N N n n N N N N N N N 5 n n n n n n n n n N d N d n n n n n n n n n n n N d N d n n n n n n n 4 n f f f f f f f f N N N N N n n n f f f f f f f f f f f f N N N n n N N N N N N N N n n n n n N N N N 3 n n n n n n n n n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n sf sf sf sf sf sf sf sf sf s s sf sf sf sf sf s s s s s s s s s s s s s s s 2 f f f f f f f f f f f f f N N N N N N N N N N d N d N N N N N N N N N N NN d N d N N N N N N N 1 s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n-’ d 5 o Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z m 3.87 01 2345678901 234567 83 # 889 S80 S91 S92 S93 S94 S95 S96 S9 7 S 98 S99 S90 S1 0 S1 0 S1 0 8 - S1 0 S1 0 S1 0 S1 0 S1 0 S1 0 S1 1 S1 1 1 1 1 1 1 1 1 87 S1S1S1S1S1S1S1S1S 91- A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 1684Aty. Dkt. No.051058-000104WOPT -’ d 3 o m 32 22 12 02 91 81 71 61 51 n n n n n n n n n n n n n n n n n 4 f f f f f f f f f f f f f f f f 1 N N N N NN d N N N N N N N N N N N 31 n n n n n n n n n n n n n n n n n 2 n n f f N d N f f f f 1 N N d n n n N N n n n n N N 11 n n n n n n n n n n n n n n n n n 0 n n n n n n f f f f 1 n n n N N N N n n n n 9 n n n n n n n f f N N n n n n n n n n 8 f f n n n n f f f f f f f N N n n N N N N N N N n n 7 n n n n N d N d n n n n n n n n n n n 6 f f n f f f f f f f f f N N n n n N N N N N N N N N n n 5 n n n n N d N d n n n n n n n n n n n 4 f f f f N N N n f f f f f f f f N n n n n N N N N N N N N 3 s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n sf sf sf sf s s sf sf sf sf sf sf sf s s s s 2 f f f f N N N NN d N d N N N N N N N N N N N 1 s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n -’ d 5 o Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z m 3.87 901 83 1 222345678901 2345 -8 # 1S1S1 2 S1 2 S1 2 S1 2 S1 2 S1 2 S1 2 S1 2 S1 3 S1 3 S1 3 S1 3 S1 3 S1 3 S1 7 S 91- A A A A A A A A A A A A A A A A A 1684Aty. Dkt. No.051058-000104WOPT

[0222] In one embodiment of any one of AS58-AS135, the nucleotide at one of positions 5-8, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G), such as : (Ngn) – a glycol nucleic acid, S-isomer; (N2p) – a 2′-phosphate nucleotide (i.e., a 3’-RNA connected by 3’-5’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively); (Tna) – a threose nucleotide (connected by 3’-3’ and 2’-5’ internucleotide linkages on the 5’ and 3’ directions, respectively); (MM) a nucleobase mismatch to the sense strand; or (Nul) an unlocked nucleic acid.

[0223] In one embodiment of any one of AS58-AS135, the nucleotide position 5, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS58-AS135, the nucleotide position 6, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS58-AS135, the nucleotide position 7, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G). In one embodiment of any one of AS58- AS135, the nucleotide position 8, counting from the 5’-end of the antisense strand is replaced with a thermaly destabilizing modification (G).

[0224] For example, the antisense strand may be selected from any one of AS136 - AS171 as recited in Table G. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT -’ 36 n n N N N n n n N N N n n n N N N n n n N N N n n n N N N 5 N d N d n n n n N d N d n n n n N d N d n n n n N d N d n n n n N d N d n n n 4 n n n n n f N n n n n n f N n n n n n f N n n n n n f N n n n n n 3 n n n n n n n n n n n n n n n n n n s n s n s n s n s n s n s n s n s n s n s n s sf sf sf sf sf s sf sf sf sf sf s sf sf sf sf sf s sf sf sf sf sf s sf s s s 2 N d N d N N NN d N N N N N N f f f N N N N N N N d N N N N NN d N N N N 1 s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n s n-’ d 5 o Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z Z m 3. 678901 2345678901 23 45678901 2 87 333344444444445555 555555666364 83 # 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 61 -8 SSSSSSSSSSSSSSSSSS SSSSSSSSSSS 79 A A A A A A A A A A A A A A A A A A A A A A A A A A A A A 1-1684Aty. Dkt. No.051058-000104WOPT -’ d 3 o m 32 22 1202 s n 91 n n n n n n n 81 n s n s n s n s n s n s n 71 n n n n n n n 6 f n f f f f f 1 N N N N N N 51 n n n n n n n 4 f f f f f f f 1 N N N N N N N 31 n n n n n n n 2 f N n n n f 1 N d n N 11 n n n n n n n 01 n n n n n n n 9 n n n n f f N N n 8 n n n n n f N n 7 G G G G G G G 6 n n n f f f N N N n 5 n N d N d n n n n 4 f f N n n n n n N 3 s n s n s n s n s n s n s n sf s s s s s s 2 f f f f f NN d N N N N N 1 s n s n s n s n s n s n s n -’ d 5 o Z Z Z Z Z Z Z m 3. 567890 87 66666717 83 # 1S1S1S1S1S1S1 -8 S 79 A A A A A A A 1-1684Aty. Dkt. No.051058-000104WOPT

[0225] In a further embodiment of each of the preceding exemplary sense and antisense strands, each of sense strand S1 through S123 can be hybridized or duplexed with any one of antisense strands AS1 through AS171. In other words, the longer double-stranded oligonucleotide product described herein comprises any one of the sense strands S1 through S123hybridized / duplexed with any one of the antisense strands AS1 through AS171. In certain embodiments, the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 23 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 21 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 21 nucleotides. In certain embodiments, the sense strand is selected from an embodiment having 19 nucleotides and the antisense strand is selected from an embodiment having 19 nucleotides.

[0226] In some embodiments, the longer single-stranded oligonucleotide described herein is any one of the sense strands S1 through S123 or any one of the antisense strands AS1 through AS171. Compositions

[0227] The dsRNA or oligonucleotide described herein can be formulated in compositions. For example, the dsRNA or oligonucleotide described herein can be formulated into pharmaceutical compositions for therapeutic use. Accordingly, in another aspect, provided herein is a pharmaceutical composition comprising a therapeuticalyeffective amount of one or more of the dsRNA or oligonucleotide described herein, taken alone, or formulated together with one or more pharmaceuticaly acceptable carriers (additives), excipient and / or diluents.

[0228] The pharmaceutical compositions can be specialy formulated for administration in solid or liquid form, including those adapted for the folowing: (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 controled-release patch or spray applied to the skin; (4) intravaginaly or intrarectaly, for example, as a pessary, cream or foam; (5) sublingualy; (6) ocularly; (7) transdermaly; or (8) nasaly. Delivery using subcutaneous or intravenous methods can be particularly advantageous. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0229] The phrase “therapeuticaly effective amount” as used herein means that amount of a compound, material, or composition comprising a conjugate described herein which is effective for producing some desired therapeutic effect in at least a sub-population of cels in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.

[0230] The phrase “pharmaceuticaly 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, alergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0231] As used herein, a “pharmaceuticaly acceptable carrier” is intended to include any and al solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceuticaly active substances is wel known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceuticaly active substances is known in the art.

[0232] Pharmaceutical compositions for use with the methods described herein can be formulated in a conventional manner using one or more physiologicaly acceptable carriers or excipients. For example, a dsRNA or oligonucleotide described herein can be formulated for administration by, for example, by intravenous, oral, aerosol, or topical route. The compositions can be formulated for intralesional, intratumoral, intraperitoneal, subcutaneous, intramuscular, or intravenous injection, infusion, liposome-mediated delivery, topical, intrathecal, gingival pocket, per rectum, intrabronchial, nasal, transmucosal, intestinal, oral, ocular, or otic delivery.

[0233] Techniques and formulations generaly can be found in Remington’s Pharmaceutical Sciences, Meade Publishing Co., Easton, PA. For systemic administration, injection is preferred, including intramuscular, intrathecal, intravenous, intraperitoneal, and subcutaneous. For injection, a dsRNA described herein can be formulated in liquid solutions, preferably in physiologicaly compatible bufers such as Hank’s solution or Ringer’s solution. In addition, the dsRNA can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.

[0234] The dsRNA or oligonucleotide can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. LNP and liposome formulations

[0235] The dsRNAs or oligonucleotides described herein can be formulated with one or more lipids for delivery. For example, the dsRNAs or oligonucleotides described herein can be formulated in lipid particles. As used herein, the term “lipid particle” refers to a vesicle formed by one or more lipid components. Lipid particles are typicaly used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a celular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Generaly, lipid particle compositions for such delivery are composed of ionizable or cationic lipids, phospholipids (especialy compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids. The ionizable lipid is typicaly employed to condense the nucleic acid cargo at low pH and to drive membrane association and fusogenicity. The phospholipid is typicaly employed to enhance fusogenicity. The cholesterol is typicaly employed to provide membrane integrity. The PEG-lipid is typicaly employed to provide steric stabilization. The sum composition of lipids typicaly dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cel uptake properties. A lipid particle can be a lipid nanoparticle (LNP).

[0236] In some embodiments, a lipid particle described herein comprises an ionizable lipid. As used herein, the term “ionizable lipid” refers to lipids having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. Typicaly, ionizable lipids are lipids comprising at least one amino group that is positively charged or becomes protonated under acidic conditions, for example at pH of 6.5 or lower. It wil be understood by one of ordinary skil in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that al of the lipid be present in the charged or neutral form. Generaly, ionizable lipids have a pKa of the protonatable group in the range of about 4 to about 7. Ionizable lipids are also refered to as cationic lipids herein.

[0237] In some embodiments, the ionizable lipid is MC3 (6Z,9Z,28Z,31Z)-heptatriaconta- 6,9,28,31-tetraen-19-yl-4-(dimethylamino) butanoate (DLin-MC3-DMA or MC3) . In some 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT embodiments, the ionizable lipid is the lipid ATX-002. In some embodiments, the ionizable lipid is (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amin.e. In some embodiments, the ionizable lipid is Compound 6 or Compound 22 described in WO2015 / 199952, content of which is incorporated herein by reference in its entirety.

[0238] Without limitations, ionizable lipid can comprise 20-90% (mol) of the total lipid present in the lipid particle.

[0239] As used herein, the term “non-cationic lipid” refers to any amphipathic lipid as wel as any other neutral lipid or anionic lipid. Accordingly, the non-cationic lipid can be a neutral uncharged, zwiterionic, or anionic lipid. Non-cationic lipids are typicaly employed to enhance fusogenicity. Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl-phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-1- trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl- phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidicacid,cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from faty acids having C10-C24carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0240] Other examples of non-cationic lipids suitable for use in the lipid particles include nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated faty acid amides, dioctadecyldimethyl ammonium bromide, ceramide, sphingomyelin, and the like. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0241] In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In some preferred embodiments, the non-cationic lipid is DPSC.

[0242] tThe non-cationic lipid can comprise 0-30% (mol) of the total lipid present in the lipid particle. For example, the non-cationic lipid content is 5-20% (mol) or 10-15% (mol) of the total lipid present in the lipid particle. In various embodiments, the molar ratio of ionizable lipid to the neutral lipid ranges from about 2:1 to about 8:1.

[0243] In some embodiments, the lipid particle can further comprise a conjugated lipid molecule. As used herein, the term “conjugated lipid” refers to a lipid molecule conjugated with a non-lipid molecule, such as a PEG, polyoxazoline, polyamide, or polymer (e.g., cationic polymer). Generaly, these lipids are used to inhibit aggregation of lipid particles and / or provide steric stabilization. Exemplary conjugated lipids include, but are not limited to, PEG-lipid conjugates, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), cationic-polymer lipid (CPL) conjugates, and mixtures thereof. In some embodiments, the conjugated lipid molecule is a PEG-lipid conjugate, for example, a (methoxy polyethylene glycol)-conjugated lipid.

[0244] Exemplary PEG-lipid conjugates include, but are not limited to, PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-O- (2',3'-di(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) butanedioate (PEG-S- DMG)), PEG dialkoxypropylcarbam, N-(carbonyl-methoxypolyethylene glycol 2000)-1,2- distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, or a mixture thereof.

[0245] The PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. The PEG-lipid can be one or more of PEG-DMG, PEG-dilaurylglycerol, PEG-dipalmitoylglycerol, PEG- disterylglycerol, PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, PEG-disterylglycamide, PEG-cholesterol (1-[8'-(Cholest-5-en-3[beta]-oxy)carboxamido-3',6'- dioxaoctanyl] carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4- Ditetradecoxylbenzyl- [omega]-methyl-poly(ethylene glycol) ether), and 1,2-dimyristoyl-sn- glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]. In some examples, the PEG-lipid can be PEG-DMG, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000]. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0246] The PEG or the conjugated lipid can comprise 0-20% (mol) of the total lipid present in the lipid particle. In some embodiments, PEG or the conjugated lipid content is 0.5-10% or 2-5% (mol) of the total lipid present in the lipid particle.

[0247] In some embodiments, the lipid particle can further comprise a component, such as a sterol, to provide membrane integrity. One exemplary sterol that can be used in the lipid particle is cholesterol and derivatives thereof. Non-limiting examples of cholesterol derivatives include polar analogues such as 5α-cholestanol, 5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether, cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogues such as 5α- cholestane, cholestenone, 5α-cholestanone, 5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue such as cholesteryl-(4′-hydroxy)-butyl ether.

[0248] The component providing membrane integrity, such as a sterol, can comprise 0-50% (mol) of the total lipid present in the lipid particle. In some embodiments, such a component is 20- 50% (mol) 30-40% (mol) of the total lipid content of the lipid particle.

[0249] In one embodiment, the lipid particle comprises: an ionizable lipid; a non-cationic lipid; a conjugated lipid that inhibits aggregation of particles; and a sterol. Molar ratios of the ionizable lipid, non-cationic-lipid, sterol, and PEG / conjugated lipid can be varied as needed. For example, the lipid particle can comprise 30-70% ionizable lipid by mole or by total weight of the composition, 0-60% cholesterol by mole or by total weight of the composition, 0-30% non- cationic-lipid by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. Preferably, the composition comprises 30-40% ionizable lipid by mole or by total weight of the composition, 40-50% cholesterol by mole or by total weight of the composition, and 10-20% non-cationic-lipid by mole or by total weight of the composition. In some other embodiments, the composition is 50-75% ionizable lipid by mole or by total weight of the composition, 20-40% cholesterol by mole or by total weight of the composition, and 5 to 10% non-cationic-lipid, by mole or by total weight of the composition and 1-10% conjugated lipid by mole or by total weight of the composition. The composition may contain 60-70% ionizable lipid by mole or by total weight of the composition, 25-35% cholesterol by mole or by total weight of the composition, and 5-10% non-cationic-lipid by mole or by total weight of the composition. The composition may also contain up to 90% ionizable lipid by mole or by total weight of the composition and 2 to 15% non-cationic lipid by mole or by total weight of the composition. The formulation may also be a lipid particle formulation, for example comprising 8-30% ionizable lipid by mole or by total weight of the composition, 5-30% non-cationic lipid by mole or by total weight of the composition, and 0-20% cholesterol by mole or by total weight of the composition; 4-25% ionizable lipid by mole or by total weight of the composition, 4-25% non-cationic lipid by mole or 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT by total weight of the composition, 2 to 25% cholesterol by mole or by total weight of the composition, 10 to 35% conjugate lipid by mole or by total weight of the composition, and 5% cholesterol by mole or by total weight of the composition; or 2-30% ionizable lipid by mole or by total weight of the composition, 2-30% non-cationic lipid by mole or by total weight of the composition, 1 to 15% cholesterol by mole or by total weight of the composition, 2 to 35% conjugate lipid by mole or by total weight of the composition, and 1-20% cholesterol by mole or by total weight of the composition; or even up to 90% ionizable lipid by mole or by total weight of the composition and 2-10% non-cationic lipids by mole or by total weight of the composition, or even 100% cationic lipid by mole or by total weight of the composition. In some embodiments, the lipid particle formulation comprises ionizable lipid, phospholipid, cholesterol and a PEG-ylated lipid in a molar ratio of 50:10:38.5:1.5. In some other embodiments, the lipid particle formulation comprises ionizable lipid, cholesterol and a PEG-ylated lipid in a molar ratio of 60:38.5:1.5.

[0250] In one embodiment, the lipid particle comprises: an ionizable lipid in an amount from about 20 mol % to about 90 mol % of the total lipid present in the particle; a non-cationic lipid in an amount from about 5 mol % to about 30 mol % of the total lipid present in the particle; a conjugated lipid that inhibits aggregation of particles in an amount from about 0.5 mol % to about 20 mol % of the total lipid present in the particle; and a sterol in an amount from about 20 mol % to about 50 mol % of the total lipid present in the particle. In some embodiments, the lipid particle comprises ionizable lipid / non-cationic-lipid / sterol / conjugated lipid at a molar ratio of 50:10:38.5:1.5.

[0251] In one embodiment, the total lipid to nucleic acid (mass or weight) ratio is from about 10:1 to about 30:1. The amounts of lipids and nucleic acid can be adjusted to provide a desired N / P ratio, for example, N / P ratio of 3, 4, 5, 6, 7, 8, 9, 10 or higher. Administering to a subject

[0252] The dsRNA and / or oligonucleotide described herein may be formulated for administration in any convenient way for use in medicine, e.g., human or veterinary medicine, by analogy with other pharmaceuticals.

[0253] The dsRNA and / or oligonucleotide described herein or a pharmaceutical composition comprising same can be administered to a subject using different routes of delivery. Exemplary routes of delivery include, but are not limited to intravenous, subcutaneous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

[0254] The dsRNA and / or oligonucleotide described herein can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral, topical (including ophthalmic, vaginal, rectal, intranasal, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT transdermal), or oral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

[0255] The route and site of administration may be chosen to enhance targeting. For example, to target muscle cels, intramuscular injection into the muscles of interest would be a logical choice. Lung cels might be targeted by administering the dsRNA and / or oligonucleotide described herein in aerosol form. The vascular endothelial cels could be targeted by coating a baloon catheter with the dsRNA and / or oligonucleotide described herein and mechanicaly introducing the dsRNA and / or oligonucleotide described herein.

[0256] In one aspect, provided herein is a method of administering an dsRNA and / or oligonucleotide described herein, to a subject (e.g., a human subject). In another aspect, the present invention relates to an dsRNA and / or oligonucleotide 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 dsRNA and / or oligonucleotide described herein.

[0257] The defined amount can be an amount efective 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.

[0258] 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.

[0259] In some embodiments, the effective dose is administered with other traditional therapeutic modalities.

[0260] The dsRNA and / or oligonucleotide described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

[0261] In some embodiments, the administration of the dsRNA and / or oligonucleotide 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.

[0262] In some embodiments, the dsRNA and / or oligonucleotide described herein is administrated via subcutaneous or intravenous administration. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0263] In some embodiments, the dsRNA and / or oligonucleotide described herein is administrated via intrathecal administration.

[0264] In some embodiments, the dsRNA and / or oligonucleotide described herein is administrated via intravitreal administration. Cels

[0265] The disclosure also provides a cel comprising a compound, dsRNA or oligonucleotide described herein described herein. As used herein, the term “cel” refers to a single cel as wel as to a population of (i.e., more than one) cels. A cel can be a prokaryotic or eukaryotic cel. Exemplary cels include, but are not limited to, bacterial cels, yeast cels, plant cel, animal (including insect) or human cels. In some embodiments, the cel is a eukaryotic cel. For example, the cel is a mammalian cel. It is noted a cel can be in vivo, in vitro or ex vivo. Kits

[0266] A dsRNA or oligonucleotide described herein can be provided in a kit, e.g., as a component of a kit. For example, the kit includes (a) a dsRNA or oligonucleotide described herein, and optionaly (b) informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and / or the use of a dsRNA or oligonucleotide described herein for the methods described herein. The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the dsRNAs or oligonucleotides, their molecular weight, concentration, date of expiration, batch, or production site information, and so forth. In some embodiments, the informational material relates to using dsRNA or oligonucleotide to treat, prevent, or diagnosis of disorders and conditions.

[0267] In some embodiments, the informational material can include instructions to administer the dsRNA or oligonucleotide in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). In another embodiment, the informational material can include instructions to administer the dsRNA or oligonucleotide to a suitable subject, e.g., a human, e.g., a human having, or at risk for, a disorder or condition needing treatment.

[0268] The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in print but can also be in other formats, such as computer readable material.

[0269] Components of the kit, e.g., the dsRNA or oligonucleotide can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that the dsRNA or oligonucleotide be 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT substantialy pure and / or sterile. When the dsRNA or oligonucleotide is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When the dsRNA or oligonucleotide is provided as a dried form, reconstitution generaly is by the addition of a suitable solvent. The solvent, e.g., sterile water or bufer, can optionaly be provided in the kit.

[0270] The kit can include one or more containers for the components of the kit. In some embodiments, the kit contains separate containers, dividers, or compartments for the different components of the kit. For example, the dsRNA or oligonucleotide can be contained in a botle, vial, or syringe, and the informational material can be contained association with the container. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the dsRNA or oligonucleotide is contained in a botle, vial or syringe that has atached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more-unit dosage forms of the dsRNA or oligonucleotide. For example, the kit includes a plurality of syringes, ampules, foil packets, or blister packs, each containing a single unit dose of the dsRNA or oligonucleotide. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0271] The kit optionaly includes a device suitable for administration of the dsRNA or oligonucleotide, e.g., a syringe, inhalant, dropper (e.g., eye dropper), swab (e.g., a coton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses metered doses of the dsRNA or oligonucleotide. The disclosure also features a method of providing a kit, e.g., by combining components described herein.

[0272] In some embodiments, the kit can further comprise additional components and / or reagents for practicing the methods described herein using the dsRNA or oligonucleotide described herein. Methods of inhibiting expression of a target gene

[0273] Aspects of the disclosure also relate to methods for inhibiting the expression of a target gene in a subject. The method comprises administering to the subject in an amount suficient to inhibit expression of the target gene: (i) a double-stranded RNA described herein, where the wherein the antisense strand is substantialy complementary to a target gene; and / or (i) an oligonucleotide described herein, wherein the oligonucleotide is substantialy complementary to a target gene.

[0274] The disclosure further relates to a use of dsRNA and / or oligonucleotide for inhibiting expression of a target gene in a target cel. The disclosure further relates to a use of an 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT oligonucleotide and / or dsRNA molecule described herein for inhibiting expression of a target gene in a target cel in vitro.

[0275] Another aspect the invention relates to a method of modulating the expression of a target gene in a cel, comprising administering to said cel a dsRNA and / or oligonucleotide described herein. It is noted that administering to the cel can be in vitro or in-vivo. Methods for administering a compound to a cel are wel known and available to one of skil in the art. As used herein, administering the compound to the cel means contacting the cel with the compound so that the compound is taken up by the cel. Generaly, the cel can be contacted with the dsRNA / oligonucleotide in a cel culture e.g., in vitro or ex vivo, or the compound can be administrated to a subject, e.g., in vivo. The term “contacting” or “contact” as used herein in connection with contacting a cel includes subjecting the cels to an appropriate culture media, which comprises a dsRNA and / or oligonucleotide described herein. Where the cel is in vivo, “contacting” or “contact” includes administering the dsRNA and / or oligonucleotide described herein, e.g., in a pharmaceutical composition to a subject via an appropriate administration route such that the compound contacts the cel in vivo. For example, when the cel is in vitro, said administering to the cel can include subjecting the cel to an appropriate culture media which comprises the dsRNA / oligonucleotide. Where the cel is in vivo, said administering to the cel includes administering the dsRNA / oligonucleotide to a subject via an appropriate administration route such that the dsRNA / oligonucleotide is administered to the cel in vivo.

[0276] In some embodiments, the target gene is selected from the group consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKK gene, JNK gene, RAF gene, Erk1 / 2 gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, hepcidin, Activated Protein C, Cyclin D gene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2 / Neu gene, topoisomerase I gene, topoisomerase II alpha gene, mutations in the p73 gene, mutations in the p21(WAF1 / CIP1) gene, mutations in the p27(KIP1) gene, mutations in the PPM1D gene, mutations in the RAS gene, mutations in the caveolin I gene, mutations in the MIB I gene, mutations in the MTAI gene, mutations in the M68 gene, mutations in tumor suppressor genes, and mutations in the p53 tumor suppressor gene.

[0277] In other embodiments, the target gene is selected from the group consisting of APP, ATXN2, C9orf72, TARDBP, MAPT(Tau), HTT, SNCA, FUS, ATXN3, ATXN1, SCA1, SCA7, SCA8, MeCP2, PRNP, SOD1, DMPK, TTR, SCN9A, LRRK2, GPR75, APOE, SCD5, ELOVL1, FLNA, ALK, CHI3L1(YKL-40), RPS25, α2-AR, and GSK3α. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0278] In one embodiment, the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Colagen Type XII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), alpha-glucosidase (GAA); adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin I receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium / calmodulin dependent protein kinase I delta (CAMK2D); or Phosphodiesterase 1 (PDE1).

[0279] In one embodiment, the target gene is selected from the group consisting of myostatin (MSTN); Cholinergic Receptor Nicotinic Alpha 1 Subunit (CHRNA1); Cholinergic Receptor Nicotinic Beta 1 Subunit (CHRNB1); Cholinergic Receptor Nicotinic Delta Subunit (CHRND); Cholinergic Receptor Nicotinic Epsilon Subunit (CHRNE); Cholinergic Receptor Nicotinic Gamma Subunit (CHRNG); Colagen Type XII Alpha 1 Chain (COL13A1); Docking Protein 7 (DOK7); LDL Receptor Related Protein 4 (LRP4); Muscle Associated Receptor Tyrosine Kinase (MUSK); Receptor Associated Protein Of The Synapse (RAPSN); Sodium Voltage-Gated Channel Alpha Subunit 4 (SCN4A); and Double Homeobox 4 (DUX4), dystrophy myotonic protein kinase (DMPK), glycogen synthase 1 (GYS1), survival of motor neuron 1 (SMN1), and alpha-glucosidase (GAA).

[0280] In one embodiment, the target gene is selected from the group consisting of adrenoceptor beta 1 (ADRB1); calcium voltage-gated channel subunit alpha1 C (CACNA1C); calcium voltage-gated channel subunit alpha1 G (CACNA1G) (T type calcium cchannel); angiotensin II receptor type 1(AGTR1); Sodium Voltage-Gated Channel Alpha Subunit 2 (SCN2A); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 1 (HCN1); Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 4 (HCN4); 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT Hyperpolarization Activated Cyclic Nucleotide Gated Potassium Channel 3 (HCN3); Potassium Voltage-Gated Channel Subfamily A Member 5 (KCNA5); Potassium Inwardly Rectifying Channel Subfamily J Member 3 (KCNJ3); Potassium Inwardly Rectifying Channel Subfamily J Member 4 (KCNJ4); phospholamban (PLN); calcium / calmodulin dependent protein kinase I delta (CAMK2D); or Phosphodiesterase 1 (PDE1).

[0281] In one embodiment, the target gene is selected from the group consisting of MUC5B, TSLP, IL33, ALOX15, AGER(RAGE),MUC5AC, and STAT6.

[0282] In one embodiment, the target gene is selected from the group consisting of Delta 4- Desaturase, Sphingolipid 1 (DEGS1); leptin; foliculin (FLCN); Zinc Finger Protein 423 (ZFP423); Cyclin Dependent Kinase 6 (CDK6); Regulatory Associated Protein Of MTOR Complex 1 (RPTOR); Mechanistic Target Of Rapamycin Kinase, (mTOR); Forkhead Box P1 (FOXP1); Phosphodiesterase 3B (PDE3B); and Activin A Receptor Type 1C (ACVR1C).

[0400] In one embodiment, the target gene is selected from the group consisting of TTR for hATTR (CNS, ocular and systemic), myocilin (MYOC), Ras homolog family member A (RhoA), SSB (smal RNA binding exonuclease protection factor La), optineurin, Carbonic Anhydrase 2 (CA2), Rho associated coiled-coil containing protein kinase 1 (ROCK1), Rho associated coiled- coil containing protein kinase 2 (ROCK2), Angiopoietin-Like 7 (ANGPTL7), and cytochrome P4501B1 (CYP1B1).

[0401] In another embodiment, the target gene is selected from the group consisting of PPARG, ADIPOQ, CD36, LPL, ADAMTS9, RASD1, GYS2, CAT, DPYS, MLXIPL, VEGFA, HLA- DQA1, LIPA, CTSC, FCGR2A, GBE1, SH2B3, CTSK, CDKN2B, ELN, ARG1, HHEX, TCF7L2, CYP2A6, ALDH2, ACADS, GLYCTK, LDLR, HAL, ACER3, SLC7A7. PTPN22, CDKN1C, LEPR, SNAI2, PGM1, IGF2BP2, TTPA, ATP7B, ASPA, ADRB3, MAN2B1, RCAN1, PIGL, TBX1, LMNB1, FBP1, ETFA, LMNA, LAT2, PRKAG2, SELENBP1, TKT, PCSK1, PSAP, NDN, ACY1, SATB2, CYP21A2, POMC, CDC73, CTSH, CFTR, CTSA, G6PD, EXT1, EXT2, CPT1A, SEMA5A, WFS1, KIT, ACAT1, GGCX, FKBP6, PPARGC1B, DGCR6, HMGCS2, PEPD, WRN, LCAT, KLF13, SLC16A2, DHCR7, ITPR3, CLDN4, FZD9, SLC30A2, APOA5, HADHA, CDKAL1, PTPN2, LIPC, CD226, PON1, MCCC1, EIF2AK3, GYG1, BCL7B, AGL, VKORC1, BAZ1B, NAGS, ASL, STAR, ACP2, POLG, GAA, ALDH3A2, MANBA, ARSA, AGA, CYP27B1, CPS1, DLAT, DCXR, EIF4H, DYRK1A, GTF2I, LAMP2, CTH, EPO, FLAD1, AKT2, WAC, GLB1, RFC2, BACH2, D2HGDH, GHRL, TBL2, RRM2B, PRKACA, DLD, NEU1, ADSL, SLC22A5, ADCY10, INSR, HSD17B10, DGCR8, NPAP1, OXCT1, SDC3, HMGCL, PGAP1, MCCC2, LMF1, PIGM, UCP3, PAH, VPS33A, BCS1L, PDP1, AHCY, ALDH18A1, ENO3, MTTP, MAT1A, GNPTAB, PHGDH, BCAT2, CBS, HDAC4, LIG3, PSAT1, HGD, CTNND2, PDHB, PDHA1, NADK2, UPB1, PKLR, BCKDK, MEN1, GALT, LIMK1, SLC39A4, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT KCNJ11, PDHX, ACAD8, GSS, CHRNA7, SLC6A9, ERBB3, GLUD1, GSR, OAT, SLC6A8, CLIP2, STX1A, CARTPT, SLC25A15, DGCR2, LIPT2, NR5A1, DNM1L, PHEX, SLC30A9, B3GAT3, SLC34A3, SLC12A3, EPX, SARS2, CAPN10, ASNS, ALDOB, AGRP, MFF, GK, APOC2, CLDN3, HPRT1, PFKM, AMACR, SNRPN, HNF1B, L2HGDH, SORD, IDH2, TPMT, CYP2C19, TERT, MC4R, TMPRSS15, SLCO1B3, FGF23, PAX4, SLC30A8, MTNR1B, SI, SLCO1B1, and NR0B2. Reactive phosphorous group

[0283] Embodiments of the various aspects described herein include a reactive phosphorus group. Without wishing to be bound by a theory, reactive phosphorus groups are useful for forming internucleoside linkages including for example phosphodiester and phosphorothioate internucleoside linkages. Such reactive phosphorus groups are known in the art and contain phosphorus atoms in PIIor PV valence state including, but not limited to, phosphoramidite, H- phosphonate, alkyl-phosphonate, phosphate triesters and phosphorus containing chiral auxiliaries. Reactive phosphorous group in the form of phosphoramidites (PII chemistry) as reactive phosphites are a preferred reactive phosphorous group for solid phase oligonucleotide synthesis. The intermediate phosphite compounds are subsequently oxidized to the Pv state using known methods to yield phosphodiester or phosphorothioate internucleoside linkages.

[0284] In some embodiments, the reactive phosphorous group is -P(ORP1)N(RP2)2, -

[0285] In some embodiments, RP1is an optionaly substituted C P1 1-6alkyl. For example, R is a C1-6alkyl, optionaly 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. In some embodiments, RP1is a C1-6alkyl, optionaly substituted with a CN or –SC(O)Ph. For example, RP1is cyanoethyl (-CH2CH2CN).

[0286] In some embodiments, each RP2 is independently optionaly substituted C1-6alkyl. For example, each RP2 can be independently selected from methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, pentyl or hexyl. It is noted that when two or more RP2 groups are present in the reactive phosphorous group, they can be same or diferent. Thus, in some none-limiting examples, when 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT two or more RP2 groups are present, the RP2 groups are diferent. In some other non-limiting examples, when two or more RP2 groups are present, the RP2 groups are same. In some embodiments, each RP2 is isopropyl.

[0287] In some embodiments, both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrolidinyl, perhydropyrrolizinyl, 1,4- diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like, each of which can be optionaly substituted with 1, 2 or 3 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.

[0288] In some embodiments, RP1and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. Exemplary heterocyclyls include, but are not limited to, pyrolidinyl, piperazinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4-morpholyl, 4-piperazinyl, pyrolidinyl, perhydropyrrolizinyl, 1,4- diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyland the like, each of which can be optionaly substituted with 1, 2 or 3 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.

[0289] In the reactive phosphorous groups, each RP3 is independently optionaly substituted C1-30alkyl substituted C1-C30alkyl, optionaly substituted C2-C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2-C10alkynyl). For example, RP3 can be a C1-6alkyl, optionaly 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- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 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, RP3 is methyl, ethyl, propyl, isopropyl, n-butyl, iso- butyl, pentyl or hexyl, each of which can be optionaly substituted with a NH2, OH, C(O)NH2, COOH, halo, SH, or C1-C6alkoxy.

[0290] In some embodiments, the reactive phosphorous group is -P(ORP1)(N(RP2)2). For example, the reactive phosphorous group is -P(ORP1)(N(RP2)), where RP1 2 is 2-cyanoethyl (- CHCHCN) and P2 2 2 each R is isopropyl.

[0291] Some exemplary aspects of the disclosure are described by one or more of folowing numbered Embodiments:

[0292] Embodiment 1: An oligonucleotide comprising at least one nucleoside of Formula (A):(Formula A), wherein: B an optionaly modified nucleobase; XS is O, CH2, S, or NH; R22 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C4-30alkyl- ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), a ligand, a linker covalently bonded to one or more ligands or a bond to an internucleotide linkage to a subsequent nucleoside; R23 is a bond to an internucleotide linkage to a subsequent nucleoside, hydroxyl, protected hydroxyl, halogen, optionaly substituted C2-30alkynyl, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2- methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT C2-30alkenyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 8 9 4-30alkyl-ON(CH2R)(CH2R), -O- C4-30alkyl-ON(CH2R8)(CH2R9), phosphate group, a ligand, or a linker covalently bonded to one or more ligands; R24 is hydrogen, optionaly substituted C1-6alkyl, optionaly substituted C2-6alkenyl, optionaly substituted C2-6alkynyl, or optionaly substituted C1-6alkoxy; or R22and R24 taken together are 4’-C(R10R11)-Y 10 11 v -2’ or 4’-Y-C(RR)v-2’; Y is -O-, -CH2-, -CH(Me)-, -C(CH3)2-, -S-, -N(R12)-, -C(O)-, -C(S)-, -S(O)-, - S(O)2-, -OC(O)-, -C(O)O-, -N(R12)C(O)-, or -C(O)N(R12)-; R10 and R11 independently are H, optionaly substituted C1-C6alkyl, optionaly substituted C2-C6alkenyl or optionaly substituted C2-C6alkynyl; R12 is hydrogen, optionaly substituted C1-30alkyl, optionaly substituted C1- C30alkoxy, C1-4haloalkyl, optionaly substituted C2-4alkenyl, optionaly substituted C2-4alkynyl, optionaly substituted C1-30alkyl-CO2H, or a nitrogen-protecting group; v is 1, 2 or 3; and R5 is -L5-CH=CH-XP, where, L5 is a bond or C30alkylene (e 5 5 P 1- .g., L is a bond, i.e., R is -CH=CH-X), and XP is a phosphate group (e.g., a protected phosphate group); provided that one of R22 and R23 is a bond to an internucleotide linkage to a subsequent nucleoside and only one of R22 and R23 is a bond to an internucleotide linkage to a subsequent nucleoside.

[0293] Embodiment 2: The oligonucleotide of Embodiment 1, wherein XS is O or CH2.

[0294] Embodiment 3: The oligonucleotide of any one of Embodiments 1-2, wherein XS is O.

[0295] Embodiment 4: The oligonucleotide of any one of Embodiments 1-3, wherein R23 is a bond to an internucleotide linkage to a subsequent nucleoside.

[0296] Embodiment 5: The oligonucleotide of Embodiment 4, wherein R22 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C4- 30alkyl-ON(CH2R8)(CH2R9), or -O-C4-30alkyl-ON(CH2R8)(CH2R9); or R24 and R25 taken together are 4’-C(R10R11)-Y-2’ o 10 11 v r 4’-Y-C(R R)v-2’. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0297] Embodiment 6: The oligonucleotide of any one of Embodiments 4-5, wherein R22 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino; or R22 and R24 taken together are 4’-C(R10R11) 10 11 v-Y-2’ or 4’-Y-C(RR)v-2’.

[0298] Embodiment 7: The oligonucleotide of any one of Embodiments 4-6, wherein R22 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy; or R22 and R24 taken together are 4’-C(R10R11)-Y-2’ or 4’ 10 11 v -Y-C(RR)v-2’.

[0299] Embodiment 8: The oligonucleotide of any one of Embodiments 4-7. wherein R22 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0300] Embodiment 9: The oligonucleotide of any one of Embodiments 4-7, wherein R22 and R24 taken together are 4’-C(R10R11)-Y 10 11 v -2’ or 4’-Y-C(RR)v-2’.

[0301] Embodiment 10: The oligonucleotide of Embodiment 9, wherein R2 and R4 taken together are 4’-C(R10R11)v-Y-2’, wherein v is 1 or 2.

[0302] Embodiment 11: The oligonucleotide of Embodiment 9 or 10, wherein one of R10 and R11 is H and the other is independently H or optionaly substituted C1-C6alkyl.

[0303] Embodiment 12: The oligonucleotide of Embodiment 11, wherein R22 and R24 taken together are 4’-CH2-O-2’.

[0304] Embodiment 13: The oligonucleotide of any one of Embodiments 4-8, wherein R24 is H.

[0305] Embodiment 14: The oligonucleotide of any one of Embodiments 1-3, wherein R22 is a bond to an internucleotide linkage to a subsequent nucleoside.

[0306] Embodiment 15: The oligonucleotide of Embodiment 14, wherein R23 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C4- 30alkyl-ON(CH2R8)(CH2R9), or -O-C4-30alkyl-ON(CH2R8)(CH2R9).

[0307] Embodiment 16: The oligonucleotide of any one of Embodiments 14-15, wherein R23 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino.

[0308] Embodiment 17: The oligonucleotide of any one of Embodiments 14-16, wherein R23 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy; or R2 and R4 taken together are 4’-C(R10R11)-Y 10 11 v -2’ or 4’-Y-C(RR)v-2’. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0309] Embodiment 18: The oligonucleotide of any one of Embodiments 14-17, wherein R23 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0310] Embodiment 19: The oligonucleotide of any one of Embodiments 14-18, wherein R24 is H.

[0311] Embodiment 20: The oligonucleotide of any one of Embodiments 1-18, wherein the nucleotide of Formula (A) is of Formula(Formula A-VP’).

[0312] Embodiment 21: The oligonucleotide of Embodiment 20, wherein R23 is a bond to an internucleotide linkage to a subsequent nucleotide.

[0313] Embodiment 22: The oligonucleotide of Embodiment 21, wherein R22 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0314] Embodiment 23: The oligonucleotide of any one of Embodiments 20-22, wherein XP is -P(O)(ORV) V 2, wherein each R is independently H or oxygen protecting group.

[0315] Embodiment 24: The oligonucleotide of Embodiment 23, wherein each RV is independently H.

[0316] Embodiment 25: The oligonucleotide of Embodiment 20, wherein XP is -P(O)(ORV)2, R23 is a bond to an internucleotide linkage to a subsequent nucleotide, and R22is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy, and wherein each RV is independently H or oxygen protecting group.

[0317] Embodiment 26: The oligonucleotide of Embodiment 25, wherein each RV is independently H.

[0318] Embodiment 27: The oligonucleotide of Embodiment 20, wherein R22 is a bond to an internucleotide linkage to a subsequent nucleotide.

[0319] Embodiment 28: The oligonucleotide of Embodiment 27, wherein R23 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0320] Embodiment 29: The oligonucleotide of Embodiment 27 or 28, wherein XP is - P(O)(ORV)2, wherein each RV is independently H or oxygen protecting group. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0321] Embodiment 30: The oligonucleotide of Embodiment 29, wherein each RV is independently H.

[0322] Embodiment 31: The oligonucleotide of Embodiment 20, wherein XP is -P(O)(ORV)2, R22 is a bond to an internucleotide linkage to a subsequent nucleotide, and R23is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy, and wherein each RV is independently H or oxygen protecting group.

[0323] Embodiment 32: The oligonucleotide of Embodiment 31, wherein each RV is independently H.

[0324] Embodiment 33: The oligonucleotide of any one of Embodiments 1-32, wherein the oligonucleotide comprises from 3 to 50 nucleotides.

[0325] Embodiment 34: The oligonucleotide of any one of Embodiments 1-33, wherein the oligonucleotide comprises at least one ribonucleotide.

[0326] Embodiment 35: The oligonucleotide of any one of Embodiments 1-34, wherein the oligonucleotide comprises at least one 2’-deoxyribonucleotide.

[0327] Embodiment 36: The oligonucleotide of any one of Embodiments 1-35, wherein the oligonucleotide comprises at least one nucleoside with a modified or non-natural nucleobase in addition to the nucleoside of Formula (A).

[0328] Embodiment 37: The oligonucleotide of any one of Embodiments 1-36, wherein the oligonucleotide comprises at least one nucleoside with a modified ribose sugar in addition to the nucleoside of Formula (A).

[0329] Embodiment 38: The oligonucleotide of any one of Embodiments 1-37, wherein the oligonucleotide comprises at least one nucleoside comprising a group other than H or OH at the 2’- position of the ribose sugar in addition to the nucleoside of Formula (A).

[0330] Embodiment 39: The oligonucleotide of any one of Embodiments 1-38, wherein the oligonucleotide comprises at least one nucleoside with a 2’-F ribose in addition to the nucleoside of Formula (A).

[0331] Embodiment 40: The oligonucleotide of any one of Embodiments 1-39, wherein the oligonucleotide comprises at least one nucleoside with a 2’-OMe ribose in addition to the nucleoside of Formula (A).

[0332] Embodiment 41: The oligonucleotide of any one of Embodiments 1-40, wherein the oligonucleotide comprises at least one nucleoside comprising a moiety other than a ribose sugar in addition to the nucleoside of Formula (A).

[0333] Embodiment 42: The oligonucleotide of any one of Embodiments 1-41, wherein the oligonucleotide comprises at least one modified internucleotide linkage. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0334] Embodiment 43: he oligonucleotide of any one of Embodiments 1-42, wherein the internucleotide linkage to the subsequent nucleoside is a modified internucleotide linkage.

[0335] Embodiment 44: The oligonucleotide of Embodiment 43, wherein the modified internucleotide linkage is a phosphorothioate linkage.

[0336] Embodiment 45: The oligonucleotide of any one of Embodiments 1-44, wherein the oligonucleotide is atached to a solid support.

[0337] Embodiment 46: The oligonucleotide of any one of Embodiments 1-45, wherein oligonucleotide comprises at least one ligand.

[0338] Embodiment 47: The oligonucleotide of any one of Embodiments 1-46, wherein the oligonucleotide comprises at least one hydroxyl, phosphate or amino protecting group.

[0339] Embodiment 48: A double-stranded nucleic acid comprising a first oligonucleotide strand and a second oligonucleotide strand substantialy complementary to the first strand, wherein the first or second strand is an oligonucleotide of any one of Embodiments 1-47.

[0340] Embodiment 49: The double-stranded nucleic acid of Embodiment 48, wherein one of the first stand and second strand is the oligonucleotide of any one of Embodiments 1-47.

[0341] Embodiment 50: The double-stranded nucleic acid of Embodiment 48 or 50, wherein the first and second strand are independently 15 to 25 nucleotides in length.

[0342] Embodiment 51: The double-stranded nucleic acid any one of Embodiments 48-50, wherein double-stranded nucleic acid is capable of inducing RNA interference.

[0343] Embodiment 52: The double-stranded nucleic acid of Embodiment 51, wherein the double-stranded nucleic acid comprises an antisense strand and sense strand, and wherein the antisense strand is the oligonucleotide of any one of Embodiments 1-47.

[0344] Embodiment 53: The double-stranded nucleic acid of any one of Embodiments 48-52, wherein one or both strands have a 1 – 5 nucleotide overhang on its respective 5’-end or 3’-end.

[0345] Embodiment 54: The double-stranded nucleic acid of any one of Embodiments 48-53, wherein only one strand has a 2 nucleotide overhang on its 5’-end or 3’-end.

[0346] Embodiment 55: The double-stranded nucleic acid of any one of Embodiments 48-54, wherein only one strand has a 2 nucleotide overhand on its 3’-end.

[0347] Embodiment 56: A pharmaceutical composition comprising an oligonucleotide of any one of Embodiments 1-47 or dsRNA molecule of any one of Embodiments 48-55, alone or in combination with a pharmaceuticaly acceptable carrier or excipient.

[0348] Embodiment 57: A gene silencing kit containing an oligonucleotide of any one of Embodiments 1-47 or dsRNA molecule of any one of Embodiments 48-55.

[0349] Embodiment 58: A method for silencing a target gene in a cel, the method comprising a step of introducing into the cel: (i) a double-stranded RNA according to any one of Embodiments 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 48-55, wherein the antisense strand comprises a nucleotide sequence substantialy complementary to the target gene; or (i) an oligonucleotide according to any one of Embodiments 1-47, wherein the oligonucleotide comprises a nucleotide sequence substantialy complementary to the target gene.

[0350] Embodiment 59: 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 Embodiments 48-55, wherein the antisense strand comprises a nucleotide sequence substantialy complementary to the target gene; or (i) an oligonucleotide according to any one of Embodiments 1-47, wherein the oligonucleotide comprises a nucleotide sequence substantialy complementary to a target gene.

[0351] Embodiment 60: The method of Embodiment 59, wherein said administering is subcutaneous or intravenous administration.

[0352] Embodiment 61: A compound of Formula (B):(Formula B), wherein: B an optionaly modified nucleobase; XS is O, CH2, S, or NH; R2 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C4-30alkyl- ON(CH2R8)(CH2R9), -O-C4-30alkyl-ON(CH2R8)(CH2R9), phosphate group, reactive phosphorous group, a ligand, or a linker covalently bonded to one or more ligands; R3 is a reactive phosphorous group, hydroxyl, protected hydroxyl, halogen, optionaly substituted C2-30alkynyl, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 8 9 4-30alkyl-ON(CH2R)(CH2R), -O-C4-30alkyl- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT ON(CH2R8)(CH2R9), phosphate group, a ligand, or a linker covalently bonded to one or more ligands; R4 is hydrogen, optionaly substituted C1-6alkyl, optionaly substituted C2-6alkenyl, optionaly substituted C2-6alkynyl, or optionaly substituted C1-6alkoxy; or R4and R2 taken together are 4’-C(R10R11)v-Y-2’ or 4’-Y-C(R10R11)v-2’; Y is -O-, -CH2-, -CH(Me)-, -C(CH 12 3)2-, -S-, -N(R)-, -C(O)-, -C(S)-, -S(O)-, - S(O) 12 12 2-, -OC(O)-, -C(O)O-, -N(R)C(O)-, or -C(O)N(R)-; R10 and R11 independently are H, optionaly substituted C1-C6alkyl, optionaly substituted C2-C6alkenyl or optionaly substituted C2-C6alkynyl; R12 is hydrogen, optionaly substituted C1-30alkyl, optionaly substituted C1- C30alkoxy, C1-4haloalkyl, optionaly substituted C2-4alkenyl, optionaly substituted C2-4alkynyl, optionaly substituted C1-30alkyl-CO2H, or a nitrogen-protecting group; v is 1, 2 or 3; and R5 is -L5-CH=CH-XP, where, L5 is a bond or C alkylene (e.g., L5 5 P 1-30 is a bond, i.e., R is -CH=CH-X), and XP is a phosphate group (e.g., a protected phosphate group), provided that when one of R2 and R3 is a reactive phosphorous group, one and only one of R2 and R3 is a reactive phosphorous group.

[0353] Embodiment 62: The compound of any Embodiment 61, wherein XS is O or CH2.

[0354] Embodiment 63: The compound of any one of Embodiments 61-62, wherein XS is O.

[0355] Embodiment 64: The compound of any one of Embodiments 61-63, wherein R3 is a reactive phosphorous group, hydroxyl, or protected hydroxyl.

[0356] Embodiment 65: The compound of Embodiment 64, wherein R3 is a reactive phosphorous group.

[0357] Embodiment 66: The compound of any one of Embodiments Embodiment 64-65, wherein R2 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C alkyl-ON(CHR8)(CHR9), or -O-C 8 9 2 4-30 2 2 4-30alkyl-ON(CH2R)(CH2R); or R and R4 taken together are 4’-C(R10R11) 10 11 v-Y-2’ or 4’-Y-C(RR)v-2’.

[0358] Embodiment 67: The compound of any one of Embodiments 64-66, wherein R2 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino; or R2 and R4 taken together are 4’-C(R10R11) 10 11 v-Y-2’ or 4’-Y-C(RR)v-2’.

[0359] Embodiment 68: The compound of any one of Embodiments 64-67, wherein R2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy; or R2 and R4 taken together are 4’-C(R10R11) 10 11 v-Y-2’ or 4’-Y-C(RR)v-2’.

[0360] Embodiment 69: The compound of any one of Embodiments 64-68, wherein R2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0361] Embodiment 70: The compound of any one of Embodiments 64-69, wherein R2 and R4 taken together are 4’-C(R10R11) 10 11 v-Y-2’ or 4’-Y-C(RR)v-2’.

[0362] Embodiment 71: The compound of Embodiment 70, wherein R2 and R4 taken together are 4’-C(R10R11)v-Y-2’, wherein v is 1 or 2.

[0363] Embodiment 72: The compound of Embodiment 70 or 71, wherein one of R10 and R11 is H and the other is independently H or optionaly substituted C1-C6alkyl.

[0364] Embodiment 73: The compound of Embodiment 72 wherein R2 and R4 taken together are 4’-CH2-O-2’.

[0365] Embodiment 74: The compound of any one of Embodiments 64-69, wherein R4 is H.

[0366] Embodiment 75: The compound of any one of Embodiments 61-63, wherein R2 is a reactive phosphorous group, hydroxyl, or protected hydroxyl.

[0367] Embodiment 76: The compound of Embodiment 75, wherein R2 is a reactive phosphorous group.

[0368] Embodiment 77: The compound of any one of Embodiments Embodiment 75-76, wherein R3 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2-methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, optionaly substituted C1-30 alkyl, optionaly substituted C2-30alkenyl, optionaly substituted C2-30alkynyl, alkoxyalkylamine, alkoxyoxycarboxylate, amino, alkylamino, dialkylamino, 5-8 membered heterocyclyl, -O-C 8 9 8 9 4-30alkyl-ON(CH2R)(CH2R), or -O-C4-30alkyl-ON(CH2R)(CH2R).

[0369] Embodiment 78: The compound of any one of Embodiments 75-77, wherein R3 is hydroxyl, protected hydroxyl, halogen, optionaly substituted C1-30 alkoxy (e.g., methoxy, 2- methoxyethoxy), alkoxyalkyl (e.g., 2-methoxyethyl), hydrogen, amino, alkylamino, or dialkylamino.

[0370] Embodiment 79: The compound of any one of Embodiments 75-78, wherein R3 is hydrogen, hydroxyl, protected hydroxyl, fluoro, methoxy, ethoxy, or 2-methoxyethoxy; or R2 and R4 taken together are 4’-C(R10R11)-Y-2’ or 4’-Y-C(R10R11 v )v-2’.

[0371] Embodiment 80: The compound of any one of Embodiments 75-79, wherein R3 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0372] Embodiment 81: The compound of any one of Embodiments 75-80, wherein R4 is H.

[0373] Embodiment 82: The compound of Embodiment 61, wherein the compound is of FormulB-VP’).

[0374] Embodiment 83: The compound of Embodiment 82, wherein R3 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-disopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-disopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0375] Embodiment 84: The compound of Embodiment 83, wherein R2 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0376] Embodiment 85: The compound of any one of Embodiments 82-84, wherein XP is - P(O)(ORV)2, wherein each RV is independently H or oxygen protecting group.

[0377] Embodiment 86: The compound of Embodiment 85, wherein each RV is independently H.

[0378] Embodiment 87: The compound of Embodiment 82, wherein XP is -P(O)(ORV) 3 2; R is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- disopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-disopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite); and R2is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0379] Embodiment 88: The compound of Embodiment 87, wherein each RV is independently an oxygen protecting group.

[0380] Embodiment 89: The compound of Embodiment 82, wherein R2 is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N-disopropyl)]- phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-disopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite).

[0381] Embodiment 90: The compound of Embodiment 89, wherein R3 is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0382] Embodiment 91: The compound of Embodiment 89 or 90, wherein XP is -P(O)(ORV)2, wherein each RV is independently H or oxygen protecting group.

[0383] Embodiment 92: The compound of Embodiment 91, wherein each RV is independently H. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0384] Embodiment 93: The compound of Embodiment 82, wherein XP is -P(O)(ORV) 2 2; R is a reactive phosphorous group (e.g., a phosphoramidite, such as 3'-[(2-cyanoethyl)-(N,N- disopropyl)]-phosphoramidite, 3'-[(2-cyanoethyl)-(N,N-disopropyl)]-phosphoramidite, or 3'-[(ß- thiobenzoylethyl)-(1-pyrrolidinyl)]-thiophosphoramidite); and R3is hydrogen, hydroxyl, protected hydroxyl, fluoro, or methoxy.

[0385] Embodiment 94: The compound of Embodiment 93, wherein each RV is independently an oxygen protecting group.

[0386] Embodiment 95: The compound of Embodiment 1, wherein the compound is ,

[0190] In some nucleotides of Formula (A) or compounds of Formula (B), R5 is -CH=CH-XP. It is noted that when R5 is -CH=CH-XP, the double bond can be in the cis or trans configuration. Accordingly, in some embodiments of any one of the aspects described herein, R5 is -CH=CH- XP, and the double bond is in the cis configuration. In some other embodiments of any one of the aspects described herein, R5 is -CH=CH- XP, and the double bond is in the cis configuration. Hydroxyl protecting groups

[0387] Some embodiments of the various aspects described herein include a hydroxyl protecting group. Hydroxyl protecting groups include, but are not limited to, −ROP1, −N(ROP2)2, −C(=O)SROP1, -C(=O)ROP1, −CO OP1 OP2 OP2 OP1 OP2 OP1 2R , −C(=O)N(R )2, −C(=NR )R , −C(=NR )OR , −C(=NROP2)N(ROP2)2, -CH2OC(=O)ROP1, −S(=O)ROP1, −SO2ROP1, −Si(ROP1)3, −P(ROP3)2, −P(ROP3)+ X−, −P(OROP3), −P(OROP3) X−, −P(=O)(ROP1), −P OP3 3 2 3 2 (=O)(OR )2, and −P(=O)(N(ROP2)2)2; wherein each X− is a counterion; each ROP1 is 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 ROP1 groups are joined to form a 3-14 membered heteroreacticvvecyclyl or 5-14 membered heteroaryl ring; each ROP2 is hydrogen, −OH, −OROP1, −N(ROP3) OP1 2, −CN, −C(=O)R , −C(=O)N(ROP3), −COROP1 OP1 OP3 OP1 OP3 OP3 OP3 2 2 , −SO2R , −C(=NR )OR , −C(=NR )N(R )2, −SO2N(R )2, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT −SO2ROP3, −SO2OROP3, −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, 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 ROP2 groups are joined to form a 3- 14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each ROP3 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1-10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two ROP3 groups 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, ROP2 and ROP3 can be optionaly 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.

[0388] Hydroxyl protecting groups are wel known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.

[0389] Exemplary hydroxyl protecting groups include, but are not limited to, methyl, t- butyloxycarbonyl (BOC or Boc), methoxymethyl (MOM), methylthiomethyl (MTM), t- butylthiomethyl, (phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p- methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM), guaiacolmethyl (GUM), t-butoxymethyl, pivaloyloxymethyl (POM), acetyloxymethyl, (AM), siloxymethyl, 2- methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2- (trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 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), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl, 1-methyl-1-benzyloxyethyl, 1- methyl-1-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2- (phenylselenyl)ethyl, t-butyl, alyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl, benzyl 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (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, α- 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-1-yl)bis(4′,4″-dimethoxyphenyl)methyl, 1,1- bis(4- methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, 9-(9-phenyl- 10-oxo)anthryl, 1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formyle, acetyl, chloroacetyl, dichloroacetyl, trichloroacetyl, trifluoroacetyl, methoxyacetyl, triphenylmethoxyacetyl, phenoxyacetyl, p-chlorophenoxyacetyl, 3-phenylpropionyl, 4- oxopentanoyl (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 alyl 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-1-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, monosuccinoate, (E)-2- methyl-2-butenoate, o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′- tetramethylphosphorodiamidate, alkyl N-phenylcarbamate, borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate (Ts).

[0390] In some embodiments, hydroxyl 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, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT t-butyldiphenylsilyl and dimethoxytrityl wherein a more preferred hydroxyl protecting group is 4,4′-dimethoxytrityl. In some embodiments, hydroxyl protecting group is pivaloyloxymethyl.

[0391] The terms “protected hydroxyl” and “protected hydroxyl” as used herein mean a group of the formula -ORPro, wherein RPro is an oxygen protecting group as defined herein. Amine protecting groups

[0392] Some embodiments of the various aspects described herein include an amine protecting group (also referred to as an amino protecting group herein). Amine protecting groups include, but10 alkenyl, C2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl groups, where each RNP1 is 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 RNP1 groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RNP2 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2- 10 alkynyl, heteroC1-10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C SP3 6-14 aryl, and 5-14 membered heteroaryl, or two R groups 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 RNP1 and RNP2 can be optionaly 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.

[0393] Amine protecting groups are wel known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference.

[0394] Exemplary amide (e.g., -C(=O)RNP1) based amine protecting groups include, but are not limited to, formamide, acetamide, chloroacetamide, trichloroacetamide, trifluoroacetamide, phenylacetamide, 3-phenylpropanamide, picolinamide, 3-pyridylcarboxamide, N- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT benzoylphenylalanyl derivative, benzamide, p- phenylbenzamide, o-nitophenylacetamide, o- nitrophenoxyacetamide, acetoacetamide, (N′- dithiobenzyloxy acylamino)acetamide, 3-(p- hydroxylphenyl)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.

[0395] Exemplary carbamate (e.g., -C(=O)ORNP1) based amine 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- (1-adamantyl)-1-methylethyl carbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate, 1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t- BOC), 1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC), 1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc), 1-(3,5-di-t- butylphenyl)-1-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), alyl carbamate (Aloc), 1- isopropylalyl 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-(1,3-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, 1,1-dimethyl-3- (N,N- dimethylcarboxamido)propyl carbamate, 1,1-dimethylpropynyl carbamate, di(2- pyridyl)methyl carbamate, 2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT carbamate, isobutyl carbamate, isonicotinyl carbamate, p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate, 1-methylcyclohexyl carbamate, 1-methyl-1- cyclopropylmethyl carbamate, 1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate, 1- methyl-1-(p- phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethyl carbamate, 1- methyl-1-(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.

[0396] Exemplary sulfonamide (e.g., -S(=O) NP1 2R ) based amine 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), β- trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide, 4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS), benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

[0397] Additional exemplary amine 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-dimethylpyrole, N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE), 5-substituted 1,3-dimethyl-1,3,5- triazacyclohexan-2-one, 5-substituted 1,3- dibenzyl-1,3,5-triazacyclohexan-2-one, 1- substituted 3,5-dinitro-4-pyridone, N-methylamine, N- alylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine, N-(1- isopropyl-4- nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammonium salts, N-benzylamine, N- di(4- methoxyphenyl)methylamine, N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr), N- [(4-methoxyphenyl)diphenylmethyl]amine (MMTr), N-9-phenylfluorenylamine (PhF), N- 2,7- dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm), 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-1- cyclohexenyl)amine, N-borane and N-diphenylborinic acid derivative, N- [phenyl(pentNP1cylchromium- or tungsten)acyl]amine, N-copper chelate, N-zinc chelate, N- 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 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). Thiol protecting groups

[0398] Some embodiments of the various aspects described herein include a thiol protecting group. Thiol protecting groups include, but are not limited to, -RSP1, -N(RSP2) SP1 2, -C(=O)SR , - C(=O)RSP1, -CO SP1 SP2 SP2 SP1 SP2 SP1 SP2 SP2 2R , −C(=O)N(R )2, -C(=NR )R , -C(=NR )OR , -C(=NR )N(R )2, -S(=O)RSP1, -SO SP1 SP1 SP3 SP3+ − SP3 SP3+ − 2R , −Si(R )3, -P(R )2, -P(R )3 X, -P(OR )2, -P(OR )3 X, - P(=O)(RSP1)2, -P(=O)(ORSP3)2, and−P(=O)(N(RSP2)2)2, wherein: X- is a counterion; each RSP1 is 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 RSP1 groups 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), −CORSP1, −SORSP1, −C(=NRSP3)O SP1 SP3 SP3 2 2 2 R , −C(=NR )N(R )2, −SO SP3 SP3 SP3 SP1 SP3 SP3 SP3 2N(R )2, −SO2R , −SO2OR , −SOR , −C(=S)N(R )2, −C(=O)SR , −C(=S)SR , −P(=O)(RSP1), −P(=O) SP3 SP3 2 (OR )2, −P(=O)(N(R )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, C SP2 6-14 aryl, and 5-14 membered heteroaryl, or two R groups are joined to form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and each RSP3 is independently hydrogen, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10 alkynyl, heteroC1- 10 alkyl, heteroC2-10 alkenyl, heteroC2-10 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C a SP3 6-14 ryl, and 5-14 membered heteroaryl, or two R groups 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, RSP2 and RSP3 can be optionaly 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— 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT [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.

[0399] Sulfur protecting groups are wel known in the art and include those described in detail in Greene’s Protecting Groups in Organic Synthesis, P. G. M. Wuts, 5th Edition, John Wiley & Sons, 2014, incorporated herein by reference. Definitions

[0400] For convenience, certain terms employed herein, in the specification, examples and appended claims are colected herein. Unless stated otherwise, or implicit from context, the folowing terms and phrases include the meanings provided below. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired in the art to which it pertains. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Further, unless otherwise required by context, singular terms shal include pluralities and plural terms shal include the singular.

[0401] Unless defined otherwise, al technical and scientific terms used herein have the same meaning as those commonly understood to one of ordinary skil in the art to which this invention pertains. Although any known methods, devices, and materials may be used in the practice or testing of the invention, the methods, devices, and materials in this regard are described herein. Definitions of common terms in immunology and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 20th Edition, published by Merck Sharp & Dohme Corp., 2018 (ISBN 0911910190, 978-0911910421); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cel Biology and Molecular Medicine, published by Blackwel Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Werner Lutmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Alan Mowat, Casey Weaver (eds.), W. W. Norton & Company, 2016 (ISBN 0815345054, 978-0815345053); Lewin's Genes XI, published by Jones & Bartlet Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Curent Protocols in Protein Science (CPPS), John E. Coligan 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Waren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are al incorporated by reference herein in their entireties.

[0402] Further, the practice of the present invention can employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cel biology, biochemistry, and immunology, which are within the skil of the art. Such techniques are explained fuly in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al., 1989); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal Cel Culture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mulis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001).

[0403] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaler ranges may independently be included in the smaler ranges and are also encompassed within the invention, subject to any specificaly excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0404] Other than in the operating examples, or where otherwise indicated, al numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in al instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In some embodiments of the various aspects described herein, the term “about” when used in connection with percentages can mean ±5%. The term “about” is used herein to provide literal support for the exact number that it precedes, as wel as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specificaly recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specificaly recited number.

[0405] As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0406] The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0407] As used herein the term “consisting essentialy of” refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materialy affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0408] The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0409] The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

[0410] As used herein, the terms “siRNA”, and “iRNA agent” are used interchangeably to refer to agents that can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also refered to as a target gene. In general, the RNA to be silenced is an endogenous gene, exogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

[0411] As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target gene, e.g., mRNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., antisense strand of a dsRNA, where the antisense strand is 21 to 23 nucleotides in length.

[0412] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as wil be understood by the skiled person. Such conditions can, for example, be stringent conditions, where stringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C or 70°C for 12-16 hours folowed by washing. Other conditions, such as physiologicaly relevant conditions as may be encountered inside an organism, can apply. The skiled person wil be able to determine the set of 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

[0413] As used herein, the term “substantialy complementary”, with respect to a nucleotide sequence in relation to a reference nucleotide sequence means a nucleotide sequence having a percentage of identity between the substantialy complementary nucleotide sequence and the exact complementary sequence of said reference of at least at least 80%. e.g., at least 85%, at least 90%, at least 93%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% (i.e., exactly complementary). Preferably identity is assessed over a length of at least 15, e.g., at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides.

[0414] The term “off-target” and the phrase “of-target effects” refer to any instance in which an effector molecule against a given target causes an unintended affect by interacting either directly or indirectly with another target sequence, a DNA sequence or a celular protein or other moiety. For example, an “of-target effect” may occur when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and / or antisense strand of an siRNA.

[0415] The terms “decrease”, “reduced”, “reduction”, or “inhibit” are al used herein to mean a decrease by a statisticaly significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typicaly means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment or agent) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

[0416] The terms “increased”, “increase”, “enhance”, or “activate” are al used herein to mean an increase by a staticaly significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10- 100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statisticaly significant increase in such level.

[0417] As used herein, a “terminal” of a strand refers to position 1, counting from the nearest end of the strand. For example, a 5’-terminal refers to position 1, counting from the 5’-end of the strand. Similarly, a 3’-terminal refers to position 1, counting from the 3’-end of the strand.

[0418] As used herein, a “terminal region” of a strand refers to positions 1-4, e.g., positions 1, 2, 3, and 4, counting from the nearest end of the strand. For example, a 5’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 5’-end of the strand. Similarly, a 3’-terminal region refers to positions 1-4, e.g., positions 1, 2, 3 and 4 counting from the 3’-end of the strand.

[0419] For example, a 5’-terminal region for the antisense strand is positions 1, 2, 3 and 4 counting from the 5’-end of the antisense strand. A prefered 5’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 5’-end of the antisense strand. A 3’-terminal region for the antisense strand can be positions 1, 2, 3, and 4 counting from the 3’-end of the strand. A preferred 3’-terminal region for the antisense strand is positions 1, 2 and 3 counting from the 3’- end of the antisense strand.

[0420] Similarly, a 5’-terminal region for the sense strand is positions 1, 2, 3 and 4 counting from the 5’-end of the sense strand. A preferred 5’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 5’-end of the sense strand. A 3’-terminal region for the sense strand can be positions 1, 2, 3, and 4 counting from the 3’-end of the strand. A preferred 3’-terminal region for the sense strand is positions 1, 2 and 3 counting from the 3’-end of the sense strand.

[0421] As used herein, a “central region” of a strand refers to 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 strand. For example, the central region of a strand means positions 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the strand. A preferred central region for the sense strand is positions 6, 7, 8, 9, 10, 11, 12, 13, and 14, counting from the 5’-end of the sense strand. A more preferred central region for the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from the 5’-end of the sense strand. A prefered central region for the antisense strand is positions 9, 10, 11, 12, 13, 14, 1516 and 17, counting from 5’-end of the antisense strand. A more preferred central region for the antisense strand is positions 10, 11, 12, 13, 14, 15 and 16, counting from 5’- end of the antisense strand. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0422] As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cel culture, etc., rather than within an organism (e.g. animal or a plant). As used herein, the term “ex vivo” refers to cels which are removed from a living organism and cultured outside the organism (e.g., in a test tube). As used herein, the term "in vivo" refers to events that occur within an organism (e.g. animal, plant, and / or microbe).

[0423] As used herein, the term "subject" or "patient" refers to any organism to which a composition disclosed herein can be administered, e.g., for experimental, diagnostic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. Usualy the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, bufalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. Patient or subject includes any subset of the foregoing, e.g., al of the above, but excluding one or more groups or species such as humans, primates or rodents. In certain embodiments of the aspects described herein, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “patient” and “subject” are used interchangeably herein. A subject can be male or female.

[0424] Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of human diseases and disorders. In addition, compounds, compositions and methods described herein can be used to with domesticated animals and / or pets.

[0425] A subject can be one who has been previously diagnosed with or identified as sufering from or having a condition in need of treatment. Alternatively, a subject can also be one who has not been previously diagnosed. A “subject in need” of testing for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

[0426] In some embodiments, the subject is human. In another embodiment, the subject is an experimental animal or animal substitute as a disease model. The term does not denote a particular age or sex. Thus, adult and newborn subjects, as wel as fetuses, whether male or female, are intended to be covered. Examples of subjects include humans, dogs, cats, cows, goats, and mice. The term subject is further intended to include transgenic species. In some embodiments, the 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT subject can be of European ancestry. In some embodiments, the subject can be of African American ancestry. In some embodiments, the subject can be of Asian ancestry.

[0427] In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” of a composition to a human subject shal be restricted to prescribing a controled substance that a human subject wil self-administer by any technique (e.g., oraly, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject mater is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” of compositions includes both methods practiced on the human body and also the foregoing activities.

[0428] As used herein, the term “parenteral administration,” refers to administration through injection or infusion. Parenteral administration includes, but is not limited to, subcutaneous administration, intravenous administration, or intramuscular administration.

[0429] As used herein, the term “subcutaneous administration” refers to administration just below the skin. “Intravenous administration” means administration into a vein.

[0430] As used herein, the term “dose” refers to a specified quantity of a pharmaceutical agent provided in a single administration. In certain embodiments, a dose may be administered in two or more boluses, tablets, or injections. For example, in certain embodiments, where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection. In such embodiments, two or more injections may be used to achieve the desired dose. In certain embodiments, a dose may be administered in two or more injections to minimize injection site reaction in an individual.

[0431] As used herein, the term “dosage unit” refers to a form in which a pharmaceutical agent is provided. In certain embodiments, a dosage unit is a vial comprising lyophilized antisense oligonucleotide. In certain embodiments, a dosage unit is a vial comprising reconstituted antisense oligonucleotide.

[0432] By the terms “treat,” “treating” or “treatment of” (and grammatical variations thereof) it is meant that the severity of the subject’s condition is reduced, at least partialy improved or stabilized and / or that some aleviation, mitigation, decrease or stabilization in at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder.

[0433] The terms “prevent,” “preventing” and “prevention” (and grammatical variations thereof) refer to prevention and / or delay of the onset of a disease, disorder and / or a clinical symptom(s) in a subject and / or a reduction in the severity of the onset of the disease, disorder and / or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. The prevention can be complete, e.g., the total absence of the disease, disorder and / or clinical 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and / or clinical symptom(s) in the subject and / or the severity of onset is less than what would occur in the absence of the present invention.

[0434] The term “statisticaly significant” or “significantly” refers to statistical significance and generaly means a two-standard deviation (2SD) or greater difference.

[0435] A glycolic nucleicwherein B is a modified or unmodified nucleobase, and * is R, S, or racemic.

[0436] 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., C1’-C2’, C2’-C3’, C3’-C4’, C4’-O4’, or C1’-O4’) is absent and / or at least one of ribose carbons or oxygen (e.g., C1’, C2’, C3’, C4’ or O4’) are independently or in combination absent from the nucleotide. In some embodiments,are H, halogen, OR3, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar.

[0437] Unlocked nucleic acid (UNA) modification encompasses monomers with bonds between C1’-C4’ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1’ 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 Leters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic nucleotide can be linked via 2’-5’ or 3’-5’ linkage. In some embodiments, UNA has the4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT or unmodified nucleobase; R, R’, R”, R”’ and R”” are independently H, OH, CH3, CH2CH3, O- alkyl, NH2, NHMe or NMe2; and each * is independently R, S, or racemic. In some embodiments, the UNAmodified or unmodified nucleobase and R is H, OH or O-alkyl.

[0438] Modified unlocked nucleic acid (mUNA) modification include, but are not limited to the folowing:, wherein Base is a modified or unmodified nucleobase, and * is R, S, or racemic.

[0439] In some embodiments, the UNA modification is selected from the group consisting of: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0440] The term “abasic modification” refers to a nucleotide or analog thereof that does not have a nucleobase. Some exemplary abasic modifications include, but are not limited to, the folowing:

[0441] In some embodiments the thermaly destabilizing modification is selected from the group consisting of: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPTMod K (h'GNA) . wherein B is a modified or unmodified nucleobase, and * is R, S, or racemic.

[0442] As usedBase is a modified or unmodifed nucleobase.

[0443] As used herein TNA is a nucleotide comprising a threose sugar instead of a ribose sugar, where its 3’-position is linked to the 3’-positoon of the nucleotide upstream of it, and its 2’-psotion is linked to the 5’-positoon of the nucleotide downstream of it. TNA has theSome exemplary TNA modifications include, but are not limited to, the folowing: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT, where B is a modified or unmodified nucleobase.

[0444] As used herein, a Hyp-spacer modification comprises a hydroxyprolinol monomer, e.g.,modified or unmodified nucleobase.

[0445] Additional exemplary sugar modifications include, but are not limited to the folowing:modified or unmodified nucleobase, and R is H or C1- C6alkyl (e.g., methyl or ethyl),

[0446] Exemplary, nucleotides with impaired W-C H-bonding to complementary base on opposing strand include, but are not limited to, nucleotides comprising a nucleobase independently selected from the folowing:. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0447] Exemplary non-canonical bases with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand, include, but are not limited to, inosine, nebularine, 2-aminopurine, 2,4-difluorotoluene, 5-nitroindole, 3-nitropyrole, 4-fluoro-6- methylbenimidazole and 4-methylbenzimidazole.

[0448] Exemplary α-nucleotides include, but are not limitedmodified or unmodified nucleobase, and R is H, OH, OCH3, F, NH2, NHMe, NMe2 or O-alkyl.

[0449] Exemplary phosphate modifications known to decrease the thermal stability of dsNA duplexes compared to natural phosphodiester linkages include, but are not limited to, the folowing:, where the alkyl for the R group can be a C1-C6alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

[0450] As used herein, the term “bridged nucleic acid” includes, but is not limited to, nucleotides that comprise a five-membered or six-membered bridged structure with a fixed 3′-endo confirmation, also known as the north confirmation. The bridged structure connects the 2′-arbon (e.g., 2’-oxygen) of the ribose sugar to the 4′ carbon of the ribose sugar. Various diferent bridge structures are possible containing carbon, oxygen, nitrogen, and hydrogen atoms.

[0451] In some embodiments, the BNA is locked nucleic acid (LNA). As used herein, the term "locked nucleic acid" (LNA) generaly refers to a class of BNAs, where the ribose ring is "locked" with a methylene bridge connecting the 2′ oxygen of the ribose sugar to the 4′ carbon of the ribose sugar.

[0452] As used herein, the term “aliphatic” means a saturated or unsaturated and straight, branched, and / or cyclic hydrocarbon having the defined number of carbon atom. Examples include alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, and cycloalkylalkynyl, having the defined number of carbon atoms.

[0453] As used herein, the term “alkyl” refers to an aliphatic hydrocarbon group which can be straight or branched having 1 to about 60 carbon atoms in the chain, and which preferably have about 6 to about 50 carbons in the chain. “Lower alkyl” refers to an alkyl group having 1 to about 8 carbon atoms. “Higher alkyl” refers to an alkyl group having about 10 to about 20 carbon atoms. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT The alkyl group can be optionaly substituted with one or more alkyl group substituents which can be the same or diferent, where “alkyl group substituent” includes halo, amino, aryl, hydroxyl, alkoxy, aryloxy, alkyloxy, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy, alkoxycarbonyl, oxo and cycloalkyl. “Branched” refers to an alkyl group in which a lower alkyl group, such as methyl, ethyl or propyl, is atached to a linear alkyl chain. Exemplary alkyl groups include methyl, ethyl, propyl, i-propyl, n-butyl, t-butyl, n-pentyl, hexyl, heptyl, octyl, decyl, dodecyl, tridecyl, tetradecyl, pentadecyl and hexadecyl. Useful alkyl groups include branched or straight chain alkyl groups of 6 to 50 carbon, and also include the lower alkyl groups of 1 to about 4 carbons and the higher alkyl groups of about 12 to about 16 carbons.

[0454] A “heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms atached (e.g., a CH2 group to an NH group or an O group). The term “heteroalkyl” include optionaly substituted alkyl, alkenyl and alkynyl radicals which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus, silicon, or combinations thereof. In certain embodiments, the heteroatom(s) is placed at any interior position of the heteroalkyl group. Examples include, but are not limited to, -CH2-O-CH3, -CH2-CH2-O-CH3, -CH2-NH-CH3, - CH2-CH2-NH-CH3, -CH2-N(CH3)-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2- S-CH2-CH3, -CH2-CH2,-S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - CH2-CH=N-OCH3, and –CH=CH-N(CH3)-CH3. In some embodiments, up to two heteroatoms are consecutive, such as, by way of example, -CH2-NH-OCH3 and –CH2-O-Si(CH3)3

[0455] As used herein, the term “alkenyl” refers to an alkyl group containing at least one carbon-carbon double bond. The alkenyl group can be optionaly substituted with one or more “alkyl group substituents.” Exemplary alkenyl groups include vinyl, alyl, n-pentenyl, decenyl, dodecenyl, tetradecadienyl, heptadec-8-en-1-yl and heptadec-8,11-dien-1-yl.

[0456] As used herein, the term “alkynyl” refers to an alkyl group containing a carbon-carbon triple bond. The alkynyl group can be optionaly substituted with one or more “alkyl group substituents.” Exemplary alkynyl groups include ethynyl, propargyl, n-pentynyl, decynyl and dodecynyl. Useful alkynyl groups include the lower alkynyl groups.

[0457] As used herein, the term “cycloalkyl” refers to a non-aromatic mono- or multicyclic ring system of about 3 to about 12 carbon atoms. The cycloalkyl group can be optionaly partialy unsaturated. The cycloalkyl group can be also optionaly substituted with an aryl group substituent, oxo and / or alkylene. Representative monocyclic cycloalkyl rings include cyclopentyl, cyclohexyl and cycloheptyl. Useful multicyclic cycloalkyl rings include adamantyl, octahydronaphthyl, decalin, camphor, camphane, and noradamantyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT

[0458] “Heterocyclyl” refers to a nonaromatic 3-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Cxheterocyclyl and Cx-Cyheterocyclyl are typicaly used where X and Y indicate the number of carbon atoms in the ring system. In some embodiments, 1, 2 or 3 hydrogen atoms of each ring can be substituted by a substituent. Exemplary heterocyclyl groups include, but are not limited to piperazinyl, pyrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, piperidyl, 4- morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3- dioxanyl, 1,4-dioxanyland the like.

[0459] “Aryl” refers to an aromatic carbocyclic radical containing about 3 to about 13 carbon atoms. The aryl group can be optionaly substituted with one or more aryl group substituents, which can be the same or different, where “aryl group substituent” includes alkyl, alkenyl, alkynyl, aryl, aralkyl, hydroxyl, alkoxy, aryloxy, aralkoxy, carboxy, aroyl, halo, nitro, trihalomethyl, cyano, alkoxycarbonyl, aryloxycarbonyl, aralkoxycarbonyl, acyloxy, acylamino, aroylamino, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, rylthio, alkylthio, alkylene and —NRR', where R and R' are each independently hydrogen, alkyl, aryl and aralkyl. Exemplary aryl groups include substituted or unsubstituted phenyl and substituted or unsubstituted naphthyl.

[0460] “Heteroaryl” refers to an aromatic 3-8 membered monocyclic, 8-12 membered fused bicyclic, or 11-14 membered fused tricyclic ring system having 1-3 heteroatoms if monocyclic, 1- 6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively.

[0461] Exemplary aryl and heteroaryls include, but are not limited to, phenyl, pyridinyl, pyrimidinyl, furanyl, thienyl, imidazolyl, thiazolyl, pyrazolyl, pyridazinyl, pyrazinyl, triazinyl, tetrazolyl, indolyl, benzyl, naphthyl, anthracenyl, azulenyl, fluorenyl, indanyl, indenyl, naphthyl, tetrahydronaphthyl, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, dihydrofuro[2,3 b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl, naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl, 1,2,4- oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl, piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrolidinyl, pyrrolinyl, 2H- pyrolyl, pyrrolyl, quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl, tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl, and the like. In some embodiments, 1, 2, 3, or 4 hydrogen atoms of each ring can be substituted by a substituent.

[0462] As used herein, the term “halogen” or “halo” refers to an atom selected from fluorine, chlorine, bromine and iodine. The term “halogen radioisotope” or “halo isotope” refers to a radionuclide of an atom selected from fluorine, chlorine, bromine and iodine.

[0463] A “halogen-substituted moiety” or “halo-substituted moiety”, as an isolated group or part of a larger group, means an aliphatic, alicyclic, or aromatic moiety, as described herein, substituted by one or more “halo” atoms, as such terms are defined in this application.

[0464] The term “haloalkyl” as used herein refers to alkyl and alkoxy structures structure with at least one substituent of fluorine, chorine, bromine or iodine, or with combinations thereof. In embodiments, where more than one halogen is included in the group, the halogens are the same or they are different. The terms “fluoroalkyl” and “fluoroalkoxy” include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. Exemplary halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g. halosubstituted (C1-C3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl (CF3), perfluoroethyl, 2,2,2-trifluoroethyl, 2,2,2-trifluoro-l,l-dichloroethyl, and the like).

[0465] As used herein, the term “amino” means -NH2. The term “alkylamino” means a nitrogen moiety having one straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals atached to the nitrogen, e.g., –NH(alkyl). The term “dialkylamino” means a nitrogen moiety having at two straight or branched unsaturated aliphatic, cyclyl, or heterocyclyl radicals atached to the nitrogen, e.g., –N(alkyl)(alkyl). The term “alkylamino” includes “alkenylamino,” “alkynylamino,” “cyclylamino,” and “heterocyclylamino.” The term “arylamino” means a nitrogen moiety having at least one aryl radical atached to the nitrogen. For example, -NHaryl, and —N(aryl)2. The term “heteroarylamino” means a nitrogen moiety having at least one heteroaryl radical atached to the nitrogen. For example —NHheteroaryl, and —N(heteroaryl)2. Optionaly, two substituents together with the nitrogen can also form a ring. Unless indicated otherwise, the compounds described herein containing amino moieties can include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT tertbutoxycarbonyl, benzyloxycarbonyl, and the like. Exemplary alkylamino includes, but is not limited to, NH(C1-C10alkyl), such as —NHCH3, —NHCH2CH3, —NHCH2CH2CH3, and — NHCH(CH3)2. Exemplary dialkylamino includes, but is not limited to, —N(C1-C10alkyl)2, such as N(CH3)2, —N(CH2CH3)2, —N(CH2CH2CH3)2, and —N(CH(CH3)2)2.

[0466] The term “aminoalkyl” means an alkyl, alkenyl, and alkynyl as defined above, except where one or more substituted or unsubstituted nitrogen atoms (—N—) are positioned between carbon atoms of the alkyl, alkenyl, or alkynyl. For example, an (C2-C6) aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

[0467] The terms “hydroxyl” and “hydroxyl” mean the radical —OH.

[0468] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical atached thereto, and can be represented by one of -O-alkyl, -O- alkenyl, and -O-alkynyl. Aroxy can be represented by –O-aryl or O-heteroaryl, wherein aryl and heteroaryl are as defined herein. The alkoxy and aroxy groups can be substituted as described above for alkyl. Exemplary alkoxy groups include, but are not limited to O-methyl, O-ethyl, O-n- propyl, O-isopropyl, O-n-butyl, O-isobutyl, O-sec-butyl, O-tert-butyl, O-pentyl, O- hexyl, O- cyclopropyl, O-cyclobutyl, O-cyclopentyl, O-cyclohexyl and the like.

[0469] As used herein, the term “carbonyl” means the radical —C(O)—. It is noted that the carbonyl radical can be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, amides, esters, ketones, and the like.

[0470] As used herein, the term “oxo” means double bonded oxygen, i.e., =O.

[0471] The term “carboxy” means the radical —C(O)O—. It is noted that compounds described herein containing carboxy moieties can include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like. As used herein, a carboxy group includes –COOH, i.e., carboxyl group.

[0472] The term “ester” refers to a chemical moiety with formula -C(=O)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl and heterocycloalkyl.

[0473] The term “cyano” means the radical —CN.

[0474] The term “nitro” means the radical —NO2.

[0475] The term, “heteroatom...

Claims

Aty. Dkt. No.051058-000104WOPT CLAIMS What is claimed is:

1. A compound of Formula (I),(Formula I), or a salt thereof, wherein: B is an optionaly modified nucleobase (e.g., uracil or 5-methyluracil); X is O or S; each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); one of R2 and R3 is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C alkynyl (e.g., pr 2 3 30 2-6 opargyl); the other of R and R is -OR , wherein: R30 is hydrogen, a hydroxy protecting group, a reactive phosphorous group (e.g., a phosphoramidite), a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

2. The compound of claim 1, wherein the compound is of structure:salt thereof.

3. The compound of claim 1, wherein the compound is of structure:salt thereof. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 4. The compound of any claim 1-3, wherein each RV is independently hydrogen, optionaly, each RV is independently hydrogen.

5. The compound of any one of claims 1-3, wherein at least one RVa hydroxyl protecting group (e.g., ethyl or POM), optionaly, each RV is a hydroxyl protecting group.

6. The compound of any one of claims 1-3 or 5, wherein each RV is -CH2CH3.

7. The compound of any one of claims 1-6, wherein X is O. X ∗ V P RO 8. The compound of any one of claims 1-7, wherein ORV is selected from the group consisting of *-P(O)(OH)2, *-P(O)(OCH2CH3)2 or *-P(O)(OCH2OC(O)C(CH3)3)2, wherein * represents the bond to the remainder of the compound 9. The compound of any one of claims 1-6, wherein X is S.

10. The compound of claim 1, wherein the compound is ofor a salt thereof.

11. The compound of claim 10, wherein the compound is ofor a salt thereof.

12. The compound of claim 10, wherein the compound is ofa salt thereof.

13. The compound of any one of claims 1-12, wherein B is a modified or protected nucleobase.

14. The compound of claim 13, wherein B is a protected nucleobase comprising at least one amine or hydroxyl protecting group. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 15. The compound of any one of claims 1-14, wherein R2 is hydrogen or halogen (e.g., fluoro).

16. The compound of any one of claims 1-14, wherein R2 is -OR20.

17. The compound of claim 16, wherein R20 is optionaly substituted C1-6alkyl.

18. The compound of claim 17, wherein R20 is methyl, ethyl, propyl, 2-methoxyethyl, 1,3- dimethoxyprop-2-yl, or 2-(N-methylamino)-2-oxoethyl, optionaly, R20 is methyl or 2-(N- methylamino)-2-oxoethyl.

19. The compound of claim 16, wherein R20 is optionaly substituted C2-6alkenyl.

20. The compound of claim 16, wherein R20 is optionaly substituted C2-6alkynyl (e.g., propargyl).

21. The compound of any one of claims 15-20, wherein R3 is -OR30, wherein R30 is hydrogen, a hydroxyl protecting group, or a reactive phosphorous group.

22. The compound of claim 21, wherein R30 is hydrogen or a hydroxyl protecting group.

23. The compound of claim 22, wherein R30 is hydrogen.

24. The compound of claim 22, R30 is a hydroxyl protecting group.

25. The compound of claim 24, wherein the hydroxyl protecting group is t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS.

26. The compound of claim 21, wherein R30 is a reactive phosphorous group.

27. The compound of claim 26, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester.

28. The compound of claim 27, wherein the reactive phosphorous group is 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT N(RP2)2, -P(RP3)N(RP2)2,each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl(e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.

29. The compound of claim 28, wherein the reactive phosphorous group is -P(ORP)N(RP2)2.

30. The compound of claim 28 or 29, wherein RP is C1-6alkyl substituted with cyano or -SC(O)Ph.

31. The compound of any one of claims 28-30, wherein RP is –CH2CH2CN.

32. The compound of any one of claims 28-31, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl.

33. The compound of any one of claims 28-32, wherein each RP2 is isopropyl.

34. The compound of any one of claims 28-33, wherein RP3 is an optionaly substituted C1- C6alkyl, (e.g., methyl).

35. The compound of any one of claims 15-20, wherein R3 is -OR30, wherein R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

36. The compound of claim 35, wherein the R30 is a bond to an oligonucleotide.

37. The compound of claim 36, wherein R3 is connected to the oligonucleotide via a phosphodiester or modified internucleotide linkage (e.g., phosphorothioate). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 38. The compound of claim 36 or 37, wherein R3 is connected to the 5’-terminal of the oligonucleotide (e.g., 5’-hydroxyl at the 5’-terminal of the oligonucleotide).

39. The compound of any one of claims 1-14, wherein R3 is hydrogen or halogen (e.g., F).

40. The compound of any one of claims 1-14, wherein R3 is -OR20.

41. The compound of claim 40, wherein R20 is optionaly substituted C1-6alkyl.

42. The compound of claim 41, wherein R20 is methyl, ethyl, propyl, 2-methoxyethyl, 1,3- dimethoxyprop-2-yl, or 2-(N-methylamino)-2-oxoethyl 2-methoxyethyl, 1,3- dimethoxyprop-2-yl, or 2-(N-methylamino)-2-oxoethyl, optionaly, R20 is methyl or 2-(N- methylamino)-2-oxoethyl.

43. The compound of claim 40, wherein R20 is optionaly substituted C2-6alkenyl.

44. The compound of claim 40, wherein R20 is optionaly substituted C2-6alkenyl (e.g., propargyl).

45. The compound of any one of claims 39-44, wherein R2 is -OR30, wherein R30 is hydrogen, a hydroxyl protecting group, or a reactive phosphorous group.

46. The compound of claim 45, wherein R30 is hydrogen or a hydroxyl protecting group.

47. The compound of claim 46, wherein R30 is hydrogen.

48. The compound of claim 47, wherein R30 is a hydroxyl protecting group.

49. The compound of claim 48, wherein the hydroxyl protecting group is t-butyldimethylsilyl (TBDMS), trimethylsilyl (TMS), triethylsilyl (TES), trisopropylsilyl (TIPS), dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS), dimethylthexylsilyl, t- butyldiphenylsilyl (TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), or t-butylmethoxyphenylsilyl (TBMPS), optionaly, the hydroxyl protecting group is TBDMS.

50. The compound of claim 45, wherein R30 is a reactive phosphorous group.

51. The compound of claim 50, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 52. The compound of claim 51, wherein the reactive phosphorous group is PN(RP2), -P P3 P2 2 (R )N(R)2,each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl.

53. The compound of claim 52, wherein the reactive phosphorous group is -P(ORP)N(RP2)2.

54. The compound of claim 52 or 53, wherein RP is C1-6alkyl substituted with cyano or -SC(O)Ph.

55. The compound of any one of claims 52-54, wherein RP is –CH2CH2CN.

56. The compound of any one of claims 52-55, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl.

57. The compound of any one of claims 52-56, wherein each RP2 is isopropyl.

58. The compound of any one of claims 52-57, wherein RP3 is an optionaly substituted C1- C6alkyl, (e.g., methyl).

59. The compound of any one of claims 39-44, wherein R2 is -OR30, wherein R30 is a bond to a nucleoside or nucleotide, or a bond to an oligonucleotide.

60. The compound of claim 59, wherein R30 is a bond to an oligonucleotide.

61. The compound of claim 60, wherein R2 is connected to the oligonucleotide via a phosphodiester or modified internucleotide linkage (e.g., phosphorothioate). 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 62. The compound of claim 60 or 61, wherein R2 is connected to the 5’-terminal of the oligonucleotide (e.g., 5’-hydroxyl at the 5’-terminal of the oligonucleotide).

63. The compound of any one of claims 1-6, wherein: R2 is hydrogen, F, or -OR20, wherein R20is hydrogen, optionaly substituted C1-6alkyl, (e.g., methyl, 2- methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N-methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); and R3is -OR30, wherein R30 is hydrogen, hydroxyl protecting group, or a reactive phosphorous group.

64. The compound of claim 63, wherein R30 is hydrogen or hydroxyl protecting group.

65. The compound of claim 63, wherein R30 is a reactive phosphorous group.

66. The compound of claim 65, wherein the reactive phosphorous group is a phosphoramidite, H-phosphonate, alkyl-phosphonate, or phosphate triester.

67. The compound of claim 66, wherein the reactive phosphorous group is -P(ORP1)N(RP2), -P(SRP1)N(RP2), -P(O)(ORP1)N(RP2), -P(S)( P1 P2 2 2 2 OR )N(R )2, - P(RP3)N(RP2)2, -P(O)(SRP1)N(RP2)2, -P(O)(ORP1)H, -P(S)(ORP1)H, -P(O)(SRP1)H, -P(O)(ORP1)RP3, -P(S)(ORP1)RP3, or -P(O)(SRP1)RP3, wherein: each RP3 is an optionaly substituted C1-C30alkyl, optionaly substituted C2- C30alkenyl, or optionaly substituted C2-C30alkynyl (e.g., optionaly substituted C1-C10alkyl, optionaly substituted C2-C10alkenyl, or optionaly substituted C2- C10alkynyl); each RP1 is independently an optionaly substituted C1-6alkyl; and each RP2 is independently optionaly substituted C1-6alkyl, or both RP2 taken together with the nitrogen atom to which they are atached form an optionaly substituted 3-8 membered heterocyclyl; or RP1 and one of RP2 taken together with the atoms to which they are atached form an optionaly substituted 4-8 membered heterocyclyl. 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT 68. The compound of claim 67, wherein the reactive phosphorous group is -P(ORP1)N(RP2)2.

69. The compound of claim 67 or 68, wherein RP1 is C1-6alkyl substituted with cyano or - SC(O)Ph.

70. The compound of any one of claims 67-69, wherein RP1 is –CH2CH2CN.

71. The compound of any one of claims 67-70, wherein each RP2 is independently methyl, ethyl, propyl, or isopropyl.

72. The compound of any one of claims 67-71, wherein each RP2 is isopropyl.

73. The compound of any one of claims 66-72, wherein the reactive phosphorous group is - P(ORP1)N(RP2), where RP1 is –CH P2 2 2CH2CN, and each R is isopropyl.

74. The compound of any one of claims 63-73, wherein R2 is hydrogen, F, or -OR20, wherein R20 is methyl or 2-methoxyethyl.

75. The compound of any one of claims 63-74, wherein each RV is independently hydrogen (e.g., X ∗ V P RO ORV is *-P(O)(OH)2).

76. The compound of any one of claims 64-74, wherein each RV is independently a hydroxyl protecting group (e.g.,or *- P(O)(OCH2OC(O)C(CH3)3)2) 77. The compound of claim 1, wherein the compound is selected from the group consisting of: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT78. An oligonucleotide, having a 5’-terminal modification comprising the structure:, wherein: X is O or S; and each RV is independently hydrogen or a hydroxyl protecting group.

79. The oligonucleotide of claim 78, wherein the 5’-terminal modification comprising the structure:.

80. The oligonucleotide of claim 78, wherein the 5’-terminal modification comprising the structure:.

81. An oligonucleotide, wherein the 5’-terminal nucleotide has the structure: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT, wherein: B is an optionaly modified nucleobase (e.g., uracil); each RV is independently hydrogen or a hydroxyl protecting group (e.g., ethyl or pivaloyloxymethyl ((CH3)3CC(O)OCH2-, POM); one of R2 and R3 is hydrogen, halogen, or -OR20, wherein: R20is hydrogen, hydroxyl protecting group, optionaly substituted C1-6alkyl, (e.g., methyl, 2-methoxyethyl, 1,3-dimethoxyprop-2-yl, 2-(N- methylamino)-2-oxoethyl), optionaly substituted C2-6alkenyl, or optionaly substituted C2-6alkynyl (e.g., propargyl); the other of R2 and R3 is -OR30, wherein: R30 is a bond to an oligonucleotide (e.g., to an internucleotide linkage that connects to the subsequent nucleotide of the oligonucleotide).

82. The oligonucleotide of claim 81, wherein the 5’-terminal nucleotide has the structure:.

83. The compound of claim 81, wherein the compound is of structure:.

84. The oligonucleotide of claim 81, wherein the 5’-terminal nucleotide has the structure:.

85. The oligonucleotide of claim 84, wherein the 5’-terminal nucleotide has the structure:.

86. The oligonucleotide of claim 84, wherein the 5’-terminal nucleotide has the structure: 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT.

87. The oligonucleotide of any one of claims 79-86, wherein R3 is -OR30.

88. An oligonucleotide, wherein the oligonucleotide is a compound of any one of claims 35-38 or 59-62.

89. The oligonucleotide of any one of claims 78-88, wherein the oligonucleotide is from 10 to 50 nucleotides (e.g., from 15 to 40 nucleotides) in length, wherein the compound of Formula (I) is one nucleotide.

90. The oligonucleotide of claim 89, wherein the oligonucleotide is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length, optionaly, the oligonucleotide is 17, 18, 19, 21, 22, 23, 24 or 25 nucleotides in length.

91. The oligonucleotide of any one of claims 78-90, wherein the oligonucleotide comprises at least one) nucleic acid modification (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more independently selected modifications).

92. The oligonucleotide of claim 91, wherein the oligonucleotide comprises at least one nucleic acid modification selected from the group consisting of nucleobase modifications, sugar modifications, internucleotide linkage modifications, conjugates (e.g., ligands), and any combinations thereof.

93. The oligonucleotide of any one of claims 78-92, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-OMe nucleotides.

94. The oligonucleotide of any one of claims 78-93, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) thermaly destabilizing modification of the duplex.

95. The oligonucleotide of claim 94, wherein said thermaly destabilizing modification of the duplex is located at position 4, 5, 6, 7, or 8, counting from the 5’-end of the oligonucleotide, 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the thermaly destabilizing modification of the duplex is located at position 6, 7, or 8, counting from the 5’-end of the oligonucleotide, preferably the thermaly destabilizing modification of the duplex is located at position 7, counting from the 5’-end of the oligonucleotide.

96. The oligonucleotide of any one of claims 78-95, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-F nucleotides.

97. The oligonucleotide of claim 96, wherein the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 14 and 16, counting from the 5’-end of the oligonucleotide, preferably the oligonucleotide comprises a 2’-F nucleotide at least at positions 2, 6, 9, 14 and 16, or at least at positions 2, 6, 8, 9, 14 and 16, counting from the 5’-end of the oligonucleotide.

98. The compound of any one of claims 78-97, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2’-deoxy (2’-H) nucleotides.

99. The oligonucleotide of claim 98, wherein the oligonucleotide comprises a 2’-deoxy nucleotide at any one of positions 2, 5, 7, 12, 14 and 16, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at position 1 from the 5’-end of the oligonucleotide, optionaly, the oligonucleotide comprises a 2’-deoxy nucleotide at least at position 5, counting from the 5’-end of oligonucleotide, preferably, the oligonucleotide comprises a 2’-deoxy nucleotide at least at positions 2, 5 and 9, or at least at positions 2, 5, 7, and 12, or at least at positions 2, 5, 7, 12, 14, and 16, counting from the 5’-end of the oligonucleotide.

100. The oligonucleotide of any one of claims 78-99, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) non-natural or modified nucleobases.

101. The oligonucleotide of any one of claims 78-100, wherein the oligonucleotide comprises at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) modified internucleoside linkages.

102. The oligonucleotide of claim 78-101, wherein the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides 4861-1978-3878.3Aty. Dkt. No.051058-000104WOPT at positions 2 and 3, counting from the 5’-end of the oligonucleotide, where the compound of Formula (I) is at nucleotide position 1 from the 5’-end of the oligonucleotide; and the oligonucleotide comprises a phosphorothioate linkage between nucleotides at positions 1 and 2, and between nucleotides at positions 2 and 3, counting from the 3’-end of the oligonucleotide.

103. The oligonucleotide of any one of claims 78-102, wherein the oligonucleotide is covalently linked to a support, e.g., a solid support.

104. A double-stranded RNA (dsRNA) comprising a sense strand and an antisense strand, wherein the sense strand is substantialy complementary to the antisense strand, and wherein one of the sense or the antisense strand is an oligonucleotide of any one of claims 78-103.

105. The dsRNA of claim 104, wherein the antisense strand is the oligonucleotide of any one of claims 78-103.

106. The dsRNA of claim 104 or 105, wherein the dsRNA is capable of inducing RNA interference.

107. 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 104-106, wherein the antisense strand is substantialy complementary to a target gene; or (i) an oligonucleotide according to any one of claims 78-103, wherein the oligonucleotide is substantialy complementary to a target gene.

108. A composition comprising a compound of any one of claims 1-77, an oligonucleotide of any one of claims 78-103, or a dsRNA of any one of claims 104-106.

109. A kit comprising a compound of any one of claims 1-77, an oligonucleotide of any one of claims 78-103, or a dsRNA of any one of claims 104-106.

110. A cel comprising a compound of any one of claims 1-77, an oligonucleotide of any one of claims 78-103, or a dsRNA of any one of claims 104-106.

111. The cel of claim 110 wherein the cel is in in vivo. 4861-1978-3878.3

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