ANTISENSE COMPOUNDS HAVING ENHANCED ANTI-MICRORNA ACTIVITY
Antisense compounds, compositions and methods are provided for modulating the levels expression, processing and function of miRNAs. The antisense compounds exhibit enhanced anti-miRNA activity. Further provided are methods for enhancing the inhibitory activity of an antisense compound targeting a miRNA, comprising incorporating stability enhancing sugar modifications into the antisense compounds.
1 . An antisense compound comprising a plurality of nucleosides with substituted or unsubstituted 2′-O-alkyl modified nucleosides and a plurality of nucleosides with bicyclic sugar modified nucleosides.
2 . The antisense compound of claim 1 wherein each of said substituted or unsubstituted 2′-O-alkyl modified nucleosides have the same sugar modification and each of said bicyclic sugar modified nucleosides have the same bicyclic modification.
3 . The antisense compound of claim 1 wherein the 2′-substituent group each of said substituted or unsubstituted 2′-O-alkyl modified nucleosides is, independently, —O—(CH 2 ) j —CH 3 , —O—(CH 2 ) 2 —O—CH 3 , —O(CH 2 ) 2 —S—CH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ) or O—CH 2 —C(═O)—N(R m )(R n ), where j is 0, 1 or 2 and each R m and R n is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.
4 . The antisense compound of claim 2 wherein the wherein the bicyclic sugar of each bicyclic sugar modified nucleoside comprises a 2′-O—CH 2 -4′, or a 2′-O—(CH 2 ) 2 -4′ bridge.
5 . The antisense compound of claim 1 wherein said antisense compound comprises linked substituted or unsubstituted 2′-O-alkyl modified nucleosides having at least two internal regions of bicyclic sugar modified nucleosides wherein each internal region comprises from 1 to 4 bicyclic sugar modified nucleosides.
6 . The antisense compound of claim 5 comprising from 3 to about 7 internal regions of bicyclic sugar modified nucleosides.
7 . The antisense compound of claim 6 wherein each region of bicyclic sugar modified nucleosides is flanked on each side by from 1 to about 8 substituted or unsubstituted 2′-O-alkyl modified nucleosides.
8 . The antisense compound of claim 7 having one of the formulas:
A 5 -B 1 -A 5 -B 1 -A 4 -B 1 -A 6 , (A-A-B) 7 (-A) 2 , (A-A-A-B) 5 -A 3 , A 5 -B 1 -A 2 -B 1 -A 2 -B 1 -A 2 -B 1 -A 1 -B 1 -A 3 -B 1 -A 2 , A 3 -B 3 -A 2 -B 3 -A 2 -B 3 -A 7 , A 3 -B 3 -A 2 -B 3 -A 2 -B 3 -A 2 -B 3 -A 2 , A 3 -B 2 -A 2 -B 3 -A 2 -B 2 -A 8 , A 5 -B 2 -A 2 -B 3 -A 2 -B 3 -A 5 ,
wherein A is a substituted or unsubstituted 2′-O-alkyl modified nucleosides, B is a bicyclic sugar modified nucleoside, and each subscript number represents the number of repeats of the preceding nucleoside or block of nucleosides.
9 . The antisense compound of claim 8 wherein each 2′-substituent group of said substituted or unsubstituted 2′-O-alkyl modified nucleosides is —O—(CH 2 ) 2 —O—CH 3 and each bicyclic modified nucleoside comprises a 2′-O—CH 2 -4′ bridge.
10 . The antisense compound of claim 1 comprising from about 15 to about 30 linked nucleosides.
11 . The antisense compound of claim 1 wherein each internucleoside linking group is, independently, a phosphodiester or a phosphorothioate.
12 . The antisense compound of claim 11 further comprising a plurality of phosphorothioate internucleoside linkages.
13 . The antisense compound of claim 1 further comprising one or more regions of from 1 to 4 differentially modified nucleosides wherein said differentially modified nucleosides are different from the other nucleosides in said antisense compound.
14 . The antisense compound of claim 13 wherein said differentially modified nucleosides are 2′-deoxynucleosides.
15 . A method of enhancing the ability of a substituted or unsubstituted 2′-O-alkyl uniformly modified antisense compound to modulate the activity of a miRNA by incorporating into said antisense compound a plurality of bicyclic sugar modified nucleosides.
16 . The method of claim 15 wherein the 2′-substituent group each of said substituted or unsubstituted 2′-O-alkyl modified nucleosides is, independently, —O—(CH 2 ) j —CH 3 , —O—(CH 2 ) 2 —O—CH 3 , —O(CH 2 ) 2 —S—CH 3 , O—(CH 2 ) 2 —O—N(R m )(R n ) or O—CH 2 —C(═O)—N(R m )(R n ), where j is 0, 1 or 2 and each R m and R n is, independently, H, an amino protecting group or substituted or unsubstituted C1-C10 alkyl.
17 . The method of claim 15 wherein the bicyclic sugar of each bicyclic sugar modified nucleoside comprises a 2′-O—CH 2 -4′, or a 2′-O—(CH 2 ) 2 -4′ bridge.