Modified RNA agents with reduced off-target effect
One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. The antisense strand of the dsRNA molecule comprises at least one thermally destabilizing nucleotide occurring at a seed region; the dsRNA comprises at least four 2′-fluoro modifications, and the sense strand of the dsRNA molecule comprises ligand, wherein the ligand is an ASGPR ligand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA molecules suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA molecules, e.g., for the treatment of various disease conditions.
1. A method for silencing a target gene in a cell, the method comprising a step of introducing a dsRNA molecule into the cell, wherein the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein said antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand or a precursor thereof, wherein the antisense strand further comprises one or both of the following characteristics:
(i) 2, 3, 4, 5 or 6 2′-fluoro modifications; and
(ii) 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; and said sense strand comprises one, two or three of the following characteristics:
(i) an asialoglycoprotein receptor (ASGPR) ligand;
(ii) 2, 3, 4, or 5 2′-fluoro modifications; and
(iii) 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages.
2. A method for suppressing off-target effects caused by the antisense strand of dsRNA molecules, the method comprising a step of introducing a dsRNA molecule into a cell, wherein the dsRNA molecule comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein said antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand or a precursor thereof, wherein the antisense strand further comprises one or both of the following characteristics:
(i) 2, 3, 4, 5 or 6 2′-fluoro modifications; and
(ii) 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages; and said sense strand comprises one, two or three of the following characteristics:
(i) an asialoglycoprotein receptor (ASGPR) ligand;
(ii) 2, 3, 4, or 5 2′-fluoro modifications; and
(iii) 1, 2, 3, 4 or 5 phosphorothioate internucleotide linkages.
3. The method of claim 2 , wherein the dsRNA comprises at least four 2′-fluoro.
4. The method of claim 3 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
5. The method of claim 2 , wherein:
(i) the dsRNA molecule has the following characteristics:
a) the thermally destabilizing modification of the duplex is located in position 4-8 of the 5′ region of the antisense strand;
b) and each of the sense and antisense strands comprise at least two 2′-fluoro modifications; and
c) an ASGPR ligand attached to either end of the sense strand; or
(ii) the dsRNA molecule has the following characteristics:
a) the thermally destabilizing modification of the duplex modification is located in position 4 to 8 of the antisense strand;
b) at least two 2′-fluoro modifications;
c) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5′ end); and
d) it has a length of 18 to 35 nucleotides; or
(iii) the dsRNA molecule has the following characteristics:
a) the ASGPR ligand attached to either end of the sense strand;
b) at least two 2′-fluoro modifications; and
c) the sense strand and the antisense strand show sufficient complementarity to form a double stranded region spanning at least 19 nucleotide positions and wherein the thermally destabilizing modification of the duplex is located within said double-stranded region.
6. The method of claim 5 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
7. The method of claim 2 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of
wherein B is nucleobase, and * is R, S or racemic.
8. The method of claim 2 , wherein the thermally destabilizing modification is located in position 7 of the antisense strand.
9. The method of claim 2 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
10. The method of claim 2 , wherein the ASGPR ligand is
11. The method of claim 1 , wherein the dsRNA comprises at least four 2′-fluoro.
12. The method of claim 11 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
13. The method of claim 1 , wherein:
(i) the dsRNA molecule has the following characteristics:
a) the thermally destabilizing modification of the duplex is located in position 4-8 of the 5′ region of the antisense strand;
b) and each of the sense and antisense strands comprise at least two 2′-fluoro modifications; and
c) an ASGPR ligand attached to either end of the sense strand; or
(ii) the dsRNA molecule has the following characteristics:
a) the thermally destabilizing modification of the duplex modification is located in position 4 to 8 of the antisense strand;
b) at least two 2′-fluoro modifications;
c) phosphorothioate internucleotide linkages between nucleotide positions 1 and 2 (counting from the 5′ end); and
d) it has a length of 18 to 35 nucleotides; or
(iii) the dsRNA molecule has the following characteristics:
a) the ASGPR ligand attached to either end of the sense strand;
b) at least two 2′-fluoro modifications; and
c) the sense strand and the antisense strand show sufficient complementarity to form a double stranded region spanning at least 19 nucleotide positions and wherein the thermally destabilizing modification of the duplex is located within said double-stranded region.
14. The method of claim 13 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
15. The method of claim 1 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of
wherein B is nucleobase, and * is R, S or racemic.
16. The method of claim 1 , wherein the thermally destabilizing modification is located in position 7 of the antisense strand.
17. The method of claim 1 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
18. The method of claim 1 , wherein the ASGPR ligand is:
19. The method of claim 1 , wherein the thermally destabilizing modification of the duplex is located at position 5, 6 or 7 of the antisense strand, counting from the 5′-end of the antisense strand.
20. The method of claim 19 , wherein the thermally destabilizing modification is located in position 6 of the antisense strand, counting from the 5′-end of the antisense strand.
21. The method of claim 1 , wherein the sense strand and the antisense strand show sufficient complementarity to form a double stranded region spanning at least 19 nucleotide positions and wherein the thermally destabilizing modification of the duplex is located within said double-stranded region.
22. The method of claim 19 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of
wherein B is a nucleobase and * is R, S or racemic.
23. The method of claim 22 , wherein the thermally destabilizing modification of the duplex is
24. The method of claim 23 , wherein the thermally destabilizing modification is an (S)-GNA modification.
25. The method of claim 1 , wherein the antisense strand comprises:
(i) 2′-fluoro modifications only at positions 2, 14 and 16, counting from the 5′-end of the antisense strand; or
(ii) 2′-fluoro modifications only at positions 2, 6, 14 and 16, counting from the 5′-end of the antisense strand; or
(iii) 2′-fluoro modifications only at positions 2, 6, 8, 9, 14 and 16, counting from the 5′-end of the antisense strand.
26. The method of claim 1 , wherein the sense strand comprises a ligand.
27. The method of claim 26 , wherein the ligand is attached at the 5′-end or 3′-end of the sense strand.
28. The method of claim 27 , wherein the ligand is an asialoglycoprotein receptor (ASGPR) ligand.
29. The method of claim 28 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
30. The method of claim 29 , wherein the ASGPR ligand is:
31. The method of claim 2 , wherein the thermally destabilizing modification of the duplex is located at position 5, 6 or 7 of the antisense strand, counting from the 5′-end of the antisense strand.
32. The method of claim 31 , wherein the thermally destabilizing modification is located in position 6 of the antisense strand, counting from the 5′-end of the antisense strand.
33. The method of claim 2 , wherein the sense strand and the antisense strand show sufficient complementarity to form a double stranded region spanning at least 19 nucleotide positions and wherein the thermally destabilizing modification of the duplex is located within said double-stranded region.
34. The method of claim 31 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of
wherein B is a nucleobase and * is R, S or racemic.
35. The method of claim 34 , wherein the thermally destabilizing modification of the duplex is
36. The method of claim 35 , wherein the thermally destabilizing modification is an (S)-GNA modification.
37. The method of claim 2 , wherein the antisense strand comprises:
(i) 2′-fluoro modifications only at positions 2, 14 and 16, counting from the 5′-end of the antisense strand; or
(ii) 2′-fluoro modifications only at positions 2, 6, 14 and 16, counting from the 5′-end of the antisense strand; or
(iii) 2′-fluoro modifications only at positions 2, 6, 8, 9, 14 and 16, counting from the 5′-end of the antisense strand.
38. The method of claim 2 , wherein the sense strand comprises a ligand.
39. The method of claim 38 , wherein the ligand is attached at the 5′-end or 3′-end of the sense strand.
40. The method of claim 39 , wherein the ligand is an asialoglycoprotein receptor (ASGPR) ligand.
41. The method of claim 40 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
42. The method of claim 41 , wherein the ASGPR ligand is: