Chemically modified oligonucleotides
View Patent ↗This invention relates composition and methods for making and using chemically modified oligonucleotides agents for inhibiting gene expression.
1. An isolated oligonucleotide agent, comprising a nucleotide sequence consisting of from 12 to 23 nucleotides in length sufficiently complementary to a microRNA target sequence, wherein the nucleotide sequence of the oligonucleotide agent differs by no more than 1 or 2 nucleotides from full complementary to the microRNA target sequence, wherein the microRNA target sequence has the nucleobase sequence of miR-21, and wherein the oligonucleotide has the structure (I):
(5′) Q x Q z 1 (Q y ) n Q z 2 Q z 3 Q z 4 Q−(3′) (I)
wherein:
each Q is independently a 2′-modified nucleoside;
x, z 1 , z 2 , z 3 , and z 4 are each independently
where one of A and B is S while the other is O;
y is
n =6−17;
L is
wherein:
X is N(CO)R 7 , or NR 7 ;
each of R 1 , R 3 and R 9 , is independently, H, OH, or —CH 2 OR b , provided that at least one of R 1 , R 3 and R 9 is OH and at least one of R 1 , R 3 and R 9 is —CH 2 OR b ;
R 7 is C 1 -C 20 alkyl substituted with NR c R d or NHC(O)R d ;
R c is H or C 1 -C 6 alkyl;
R d is a carbohydrate radical; or a steroid radical, which is optionally tethered to at least one carbohydrate radical; and
R b is
where one of A and B is S while the other is O.
2. The compound of claim 1 , wherein R d is cholesterol.
3. The compound of claim 1 , wherein R 1 is —CH 2 OR b .
4. The compound of claim 3 , wherein R 9 is OH.
5. The compound of claim 4 , wherein R 1 and R 9 are trans.
6. The compound of claim 1 , wherein R 3 is OH.
7. The compound of claim 6 , wherein R 1 and R 3 are trans.
8. The compound of claim 1 , wherein R 3 is —CH 2 OR b .
9. The compound of claim 8 , wherein R 1 is OH.
10. The compound of claim 9 , wherein R 1 and R 3 are trans.
11. The compound of claim 8 , wherein R 9 is OH.
12. The compound of claim 8 , wherein R 3 and R 9 are trans.
13. The compound of claim 1 , wherein R 9 is CH 2 OR b .
14. The compound of claim 13 , wherein R 1 is OH.
15. The compound of claim 14 , wherein R 1 and R 9 are trans.
16. The compound of claim 1 , wherein X is NC(O)R 7 .
17. The compound of claim 16 , wherein R 7 is —CH 2 (CH 2 ) 3 CH 2 NHC(O)R d .
18. The compound of claim 1 , wherein each 2′-modified nucleoside is independently selected from the group consisting of a 2′-O-methoxyethyl nucleoside, a 2′-fluoro nucleoside, a 2′-O-methyl nucleoside, and an locked nucleic acid nucleoside.
19. The compound of claim 18 , wherein each 2′-modified nucleoside is a 2′-O-methoxyethyl nucleoside.
20. The compound of claim 18 , wherein each 2′-modified nucleoside is a 2′-O-methyl nucleoside.
21. The compound of claim 1 , wherein at least one nucleoside comprises a modified nucleobase.
22. The compound of claim 21 , wherein the modified nucleobase is a 5-methylcytosine.
23. An isolated oligonucleotide agent, comprising a nucleotide sequence consisting of 22 nucleotides in length, wherein the nucleotide sequence of the oligonucleotide agent is fully complementary to the nuclebase sequence of miR-21 and wherein the oligonucleotide has the structure (I):
(5′) Q x Q z 1 (Q y ) n Q z 2 Q z 3 Q z 4 Q-L (3′) (I)
wherein:
each Q is independently a 2′-O-methyl modified nucleoside;
x, z 1 , z 2 , z 3 , and z 4 are each indpendently
where one of A and B is S while the other is O;
y is
n=16;
L is
wherein:
X is N(CO)R 7 , or NR 7 ;
Each of R 1 , R 3 and R 9 , is independently, H, OH, or —CH 2 OR b provided that at least one of R 1 , R 3 and R 9 is OH and at least one of R 1 , R 3 and R 9 is —CH 2 OR b ;
R 7 is C 1 -C 20 alkyl substituted with NR c R d or NHC(O)R d ;
R c is H or C 1 -C 6 alkyl;
R d is cholesterol; and
R b is
where one of A and B is S while the other is O.
24. A method of inhibiting the activity of miR-21, comprising administering to a subject the oligonucleotide agent of claim 1 .
25. A method of inhibiting the activity or expression of miR-21 in a cell, comprising contacting the cell with the oligonucleotide agent of claim 1 .
26. The method of claim 25 , wherein the cell is a tumor cell.
27. The method of claim 26 , wherein the tumor cell is a lung tumor cell.