Compositions and methods for improving strand biased
One aspect of the present invention relates to double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene. Other aspects of the invention relate 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 double-stranded RNA (dsRNA) molecule capable of inhibiting the expression of a target gene, comprising: a sense strand and an antisense strand, each stranding having 14 to 40 nucleotides, wherein the antisense strand has sufficient complementarity to the target sequence to mediate RNA interference, wherein said sense strand, at position 1 of the 5′ end, comprises a 5′-deoxynucleoside with a morpholino at the 5′-carbon.
2. The dsRNA molecule of claim 1 , wherein said antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region, wherein said sense strand comprises an asialoglycoprotein receptor (ASGPR) ligand.
3. The dsRNA molecule according to claim 2 , wherein the dsRNA comprises at least four 2′-fluoro.
4. The dsRNA molecule according to claim 3 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
5. The dsRNA molecule according to claim 1 , further having 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.
6. The dsRNA molecule according to claim 5 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
7. The dsRNA molecule according to claim 1 , wherein the antisense strand has at least two of 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);
d) it has a length of 18 to 35 nucleotides.
8. The dsRNA molecule according to claim 7 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
9. The dsRNA molecule according to claim 1 , wherein the sense strand has at least one of the following characteristics:
a) the ASGPR ligand attached to either end of the sense strand;
b) at least two 2′-fluoro modifications;
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.
10. The dsRNA molecule according to claim 9 , wherein there are no 2′-fluoro modifications at nucleotide positions 3-9 of the antisense strand.
11. The dsRNA molecule according to claim 2 , wherein the thermally destabilizing modification of the duplex is selected from the group consisting of
wherein B is nucleobase.
12. The dsRNA molecule according to claim 2 , wherein the thermally destabilizing modification is located in position 7 of the antisense strand.
13. The dsRNA molecule according to claim 2 , wherein the ASGPR ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker.
14. The dsRNA molecule of claim 9 , wherein the ASGPR ligand is:
15. The dsRNA molecule according to claim 1 , wherein the antisense strand comprises at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region, and the dsRNA has a melting temperature of from about 40° C. to about 80° C.
16. The dsRNA of claim 1 , wherein the dsRNA further has at least one of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3 or 4 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (viii) a blunt end at 5′ end of the antisense strand; and (ix) the sense strand comprises one or more LNA modifications.
17. The dsRNA of claim 16 , wherein there are no 2′-fluoro modifications at positions 3-9 of the antisense strand.
18. The dsRNA molecule according to claim 1 , wherein the sense strand has 21 nucleotides, and the antisense strand has 23 nucleotides.
19. A method for silencing a target gene in a cell, the method comprising a step of introducing the dsRNA molecule of claim 1 into the cell.
20. A method for suppressing off-target effects caused by the sense strand of dsRNA molecules, the method comprising a step of introducing the dsRNA molecule of claim 1 into a cell.