iRNA agents with biocleavable tethers
View Patent ↗The invention relates to iRNA agents, which preferably include a monomer in which the ribose moiety has been replaced by a moiety other than ribose that further includes a tether having one or more linking groups, in which at least one of the linking groups is a cleavable linking group. The tether in turn can be connected to a selected moiety, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property. The cleavable linking group is one which is sufficiently stable outside the cell such that it allows targeting of a therapeutically beneficial amount of an iRNA agent (e.g., a single stranded or double stranded iRNA agent), coupled by way of the cleavable linking group to a targeting agent—to targets cells, but which upon entry into a target cell is cleaved to release the iRNA agent from the targeting agent.
1. A modified RNA agent comprising a first strand and optionally a second strand, wherein at least two subunits having a formula (I) is incorporated into at least one of the strands:
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 T—R d , in which T is a tether comprising one or more linking groups, wherein at least one of the linking groups is cleavable; and R d is H or a ligand;
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 H or
R b is or
wherein the Stand in each occurrence is independently the first or second strand of the iRNA agent;
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; and
n is 1-4; provided that X is N(CO)R 7 or NR 7 ; or one of R 5 , R 6 , R 11 , and R 12 is C(O)NHR 7 ; or R 14 is present.
2. The agent of claim 1 , wherein at least one of the linking groups is cleaved at least about 100 times faster under conditions chosen to mimic intracellular media than under conditions chosen to mimic extracellular media.
3. The agent of claim 1 , wherein at least one of the linking groups is cleaved at least about 50 times faster under conditions chosen to mimic intracellular media than under conditions chosen to mimic extracellular media.
4. The agent of claim 1 , wherein at least one of the linking groups is cleaved at least about 10 times faster under conditions chosen to mimic intracellular media than under conditions chosen to mimic extracellular media.
5. The agent of claim 1 , wherein at least one of the linking groups is a redox cleavable linking group, an acid cleavable linking group, an esterase cleavable linking group, a phosphatase cleavable linking group, or a peptidase cleavable linking group.
6. The agent of claim 5 , wherein at least one of the linking groups is a reductively cleavable linking group.
7. The agent of claim 6 , wherein the reductively cleavable linking group is a disulfide group.
8. The agent of claim 5 , wherein at least one of the linking groups is an acid cleavable linking group.
9. The agent of claim 8 , wherein the acid cleavable linking group is a hydrazone group.
10. The agent of claim 8 , wherein the acid cleavable linking group is an ester group.
11. The agent of claim 5 , wherein at least one of the linking groups is an esterase cleavable linking group.
12. The agent of claim 11 , wherein the esterase cleavable linking group is an ester group.
13. The agent of claim 5 , wherein at least one of the linking groups is a phosphatase cleavable linking group.
14. The agent of claim 13 , wherein the phosphatase cleavable linking group is a phosphate group.
15. The agent of claim 5 , wherein at least one of the linking groups is an peptidase cleavable linking group.
16. The agent of claim 15 , wherein the peptidase cleavable linking group is a peptide bond.
17. The agent of claim 1 , wherein R 7 has the formula R d —(E′) s -Δ-(E″) t -;
wherein:
each of E′ and E″ is a terminal linking group; and
Δ is a hydrocarbon chain that optionally includes one or more internal linking groups, G;
each of s and t is, independently, 0 or 1;
provided that one of s or t is 1, or Δ includes at least one G.
18. The agent of claim 17 , wherein each of E′, E″, and G is, independently, —NR k C(O)—, —C(O)NR k —, —OC(O)NR k —, —NR k C(O)O—, —O—, —S—, —SS—, —S(O)—, —S(O 2 )—, —NR k C(O)NR k —, —NR k C(S)NR k —, —C(O)O—, —OC(O)—, —NR k C(S)—, —NR k C(S)O—, —C(S)NR k —, —OC(S)NR k —, —NR k C(S)O—, —O—P(O)(OR k )—O—, —O—P(S)(OR k )—O—, —O—P(S)(SR k )—O—, —S—P(O)(OR k )—O—, —O—P(O)(OR k )—S—, —S—P(O)(OR k )—S—, —O—P(S)(OR k )—S—, —S—P(S)(OR k )—O—, —O—P(O)(R k )—O—, —O—P(S)(R k )—O—, —S—P(O)(R k )—O—, —S—P(S)(R k )—O—, —S—P(O)(R k )—S—, —O—P(S)(R k )—S—, —C(O)—, —NR k —R k C═NNR k —, —NHCHR k′ C(O)NHCHR k″ C(O)—, or —NHCHR k′ NHC(O)CHR k″ C(O)—; wherein
R k at each occurrence is, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, C7-C12 aralkyl; and
each of R k′ and R k″ is, independently of one another, an amino acid sidechain.
19. The agent of claim 18 , wherein at least one G is —SS—, —C(O)O—, —OC(O)—, —R k C═NNR k —, —NHCHR k′ C(O)NHCHR k″ C(O)—, or —NHCHR k′ NHC(O)CHR k″ C(O)—.
20. The agent of claim 1 , wherein R d is a lipophilic moiety; a folic acid radical; a steroid radical; a carbohydrate radical; a vitamin A radical; a vitamin E radical; or a vitamin K radical.
21. The agent of claim 20 , wherein R d is a cholesterol radical.
22. The agent of claim 1 , wherein the two subunits of formula (I) are incorporated into a sense strand.
23. The agent of claim 1 , wherein the two subunits of formula (I) are placed at two positions selected from positions 1, 2 and 3 of the 3′ end of a sense strand.
24. The agent of claim 1 , wherein three subunits having a formula (I) are incorporated into at least one of said strands.
25. The agent of claim 1 , wherein the three subunits having a formula (I) are placed at positions 1, 2 and 3 of the 3′ end of a sense strand.
26. The agent of claim 1 , wherein -(E′) s -Δ-(E″) t - is selected from the group consisting of: