Ligand-conjugated monomers
This invention relates composition and methods for making and using chemically modified oligonucleotides agents for inhibiting gene expression.
1. A method of inhibiting a target gene comprising providing a conjugate to a cell, wherein the conjugate comprises an oligonucleotide agent that can target a substantially complementary miRNA and a ligand, and wherein at least one subunit having a formula (I) is incorporated into said oligonucleotide agent:
wherein:
X is N(CO)R 7 , NR 7 or CH 2 ;
Y is NR 8 , O, S, CR 9 R 10 , or absent;
Z is CR 11 R 12 or absent;
Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , OR b , (CH 2 ) n OR a , or (CH 2 ) n OR b , provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is OR a or OR b and that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is (CH 2 ) n OR a , or (CH 2 ) n OR b ;
Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 ;
R 7 is R d ; or C 1 -C 20 alkyl substituted with NR c R d or NHC(O)R d ;
R 8 is C 1 -C 6 alkyl;
R 13 is hydroxy, C 1 -C 4 alkoxy, or halo;
R 14 is NR c R 7 ;
R a is:
R b is:
Each of A and C is, independently, O or S;
B is OH, O − , or
R c is H or C 1 -C 6 alkyl;
R d is the ligand; and
n is 1-4.
2. The method of claim 1 , wherein said conjugate is administered to an organism.
3. The method of claim 1 , wherein said cell is outside an organism.
4. The method of claim 3 , wherein said cell is a cell of a cell line.
5. A method of modulating expression of a target gene in a subject, the method comprising administering an conjugate to a subject, wherein the conjugate comprises an oligonucleotide agent that can target a substantially complementary miRNA and a ligand, wherein at least one subunit having a formula (I) is incorporated into said oligonucleotide agent:
wherein:
X is N(CO)R 7 , NR 7 or CH 2 ;
Y is NR 8 , O, S, CR 9 R 10 , or absent;
Z is CR 11 R 12 or absent;
Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , OR b , (CH 2 ) n OR a , or (CH 2 ) n OR b , provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is OR a or OR b and that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is (CH 2 ) n OR a , or (CH 2 ) n OR b ;
Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 ;
R 7 is R d ; or C 1 -C 20 alkyl substituted with NR c R d or NHC(O)R d ;
R 8 is C 1 -C 6 alkyl;
R 13 is hydroxy, C 1 -C 4 alkoxy, or halo;
R 14 is NR c R 7 ;
R a is:
R b is:
Each of A and C is, independently, O or S;
B is OH, O − , or
R c is H or C 1 -C 6 alkyl;
R d is the ligand; and
n is 1-4.
6. A method of treating a kidney, ocular, or viral disorder, said method comprising administering a conjugate to a subject, wherein the conjugate comprises an oligonucleotide agent that can target a substantially complementary miRNA and a ligand, wherein at least one subunit having a formula (I) is incorporated into said oligonucleotide agent:
wherein:
X is N(CO)R 7 , NR 7 or CH 2 ;
Y is NR 8 , O, S, CR 9 R 10 , or absent;
Z is CR 11 R 12 or absent;
Each of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is, independently, H, OR a , OR b , (CH 2 ) n OR a , or (CH 2 ) n OR b , provided that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is OR a or OR b and that at least one of R 1 , R 2 , R 3 , R 4 , R 9 , and R 10 is (CH 2 ) n OR a , or (CH 2 ) n OR b ;
Each of R 5 , R 6 , R 11 , and R 12 is, independently, H, C 1 -C 6 alkyl optionally substituted with 1-3 R 13 , or C(O)NHR 7 ; or R 5 and R 11 together are C 3 -C 8 cycloalkyl optionally substituted with R 14 ,
R 7 is R d ; or C 1 -C 20 alkyl substituted with NR c R d or NHC(O)R d ;
R 8 is C 1 -C 6 alkyl;
R 13 is hydroxy, C 1 -C 4 alkoxy, or halo;
R 14 is NR c R 7 ;
R a is:
R b is:
Each of A and C is, independently, O or S;
B is OH, O − , or
R c is H or C 1 -C 6 alkyl;
R d is the ligand; and
n is 1-4.
7. The method of claim 1 , wherein X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent.
8. The method of claim 7 , wherein R 7 is (CH 2 ) 5 NHR d or (CH 2 ) 5 NHC(O)R d .
9. The method of claim 1 , wherein the R d ligand is a steroid radical optionally tethered to at least one carbohydrate radical; a folic acid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical; or a vitamin B 12 radical.
10. The method of claim 9 , wherein the R d ligand is a carbohydrate cholesterol radical.
11. The method of claim 1 , wherein the miRNA is selected from the group consisting of miR-21, miR-16, miR-22, miR-122, miR-155, miR-192, miR-194, and miR-375.
12. The method of claim 5 , wherein X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent.
13. The method of claim 12 , wherein R 7 is (CH 2 ) 5 NHR d or (CH 2 ) 5 NHC(O)R d .
14. The method of claim 5 , wherein the R d ligand is a steroid radical optionally tethered to at least one carbohydrate radical; a folic acid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical; or a vitamin B 12 radical.
15. The method of claim 14 , wherein the R d ligand is a carbohydrate cholesterol radical.
16. The method of claim 5 , wherein the miRNA is selected from the group consisting of miR-21, miR-16, miR-22, miR-122, miR-155, miR-192, miR-194, and miR-375.
17. The method of claim 6 , wherein X is N(CO)R 7 or NR 7 , Y is CR 9 R 10 , and Z is absent.
18. The method of claim 17 , wherein R 7 is (CH 2 ) 5 NHR d or (CH 2 ) 5 NHC(O)R d .
19. The method of claim 6 , wherein the R d ligand is a steroid radical optionally tethered to at least one carbohydrate radical; a folic acid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; a vitamin K radical; or a vitamin B 12 radical.
20. The method of claim 19 , wherein the R d ligand is a carbohydrate cholesterol radical.
21. The method of claim 6 , wherein the miRNA is selected from the group consisting of miR-21, miR-16, miR-22, miR-122, miR-155, miR-192, miR-194, and miR-375.