Modified double stranded oligonucleotide
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 dsRNA agent comprising:
a sense strand having a length of 17-35 nucleotides;
an antisense strand having a length of 17-35 nucleotides;
at least two phosphorothioate internucleotide linkages between the first five nucleotides of the antisense strand, counting from the 5′ end of the antisense strand; and
only three, four, five or six 2′-deoxy modifications on the antisense strand;
wherein:
at least two 2′-deoxy modifications are in a central region of the sense strand, wherein the central region of the sense strand is positions 7, 8, 9, 10, 11, 12, and 13 counting from the 5′-end of the sense strand;
three of the 2′-deoxy modifications of the antisense strand are at positions 2, 12, and 14, counting from the 5′-end of the antisense strand;
the sense strand and the antisense strand form a duplex region containing between 17 to 25 base pairs;
the dsRNA agent comprises a ligand;
the sense strand does not comprise a glycol nucleic acid (GNA); and
the 2′-deoxy modifications are not 2′-deoxy-2′-fluoro (2′-F) nucleoside.
2 . The dsRNA agent of claim 1 , wherein the dsRNA agents have all natural nucleotides, or less than 20% non-natural nucleotides.
3 . The dsRNA agent of claim 1 , wherein the dsRNA comprises a sense strand having a length of 18-30 nucleotides.
4 . The dsRNA agent of claim 1 , wherein the dsRNA comprises an antisense strand having a length of 18-30 nucleotides.
5 . The dsRNA agent of claim 1 , wherein the dsRNA comprises an antisense strand having a length of 18-23 nucleotides, and five 2′-deoxy modifications in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand.
6 . The dsRNA agent of claim 1 , wherein at least one of the 2′-deoxy modifications is in the sense strand at position 11, counting from the 5′-end of the sense strand.
7 . The dsRNA agent of claim 1 , wherein at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.
8 . The dsRNA agent of claim 1 , wherein at least five of the 2′-deoxy modifications are in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.
9 . The dsRNA agent of claim 2 , wherein the non-natural nucleotide is selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA) nucleotides, hexitol nucleic acid (HNA) nucleotides, cyclohexenyl nucleic acid (CeNA) nucleotides, 2′-methoxyethyl nucleotides, 2′-O-allyl nucleotides, 2′-C-allyl nucleotides, 2′-fluoro nucleotides, 2′-O—N-methylacetamido (2′-O-NMA) nucleotides, 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotides, 2′-O-aminopropyl (2′-O-AP) nucleotides, and 2′-ara-F nucleotides.
10 . The dsRNA agent of claim 2 , wherein the natural nucleotide is selected from the group consisting of 2′-OH nucleotides, 2′-OMe nucleotides, and 2′-deoxy nucleotides.
11 . The dsRNA agent of claim 1 , wherein the ligand is an ASGPR ligand.
12 . The dsRNA agent of claim 1 , wherein:
the sense strand has:
a length of 19-25 nucleotides and only two 2′-deoxy modifications in a central region of the sense strand, wherein the central region of the sense strand is positions 7, 8, 9, 10, 11, 12 and 13, counting from 5′-end of the sense strand;
and
the antisense strand has:
a length of 19-25 nucleotides, and
three, four, or five 2′-deoxy modifications in the antisense strand, and wherein three of the 2′-deoxy modifications in the antisense strand are at positions 2, 12 and 14, counting from the 5′-end of the antisense strand,
and
wherein the duplex region contains between 17 to 25 base pairs.
13 . The dsRNA agent of claim 1 , wherein the dsRNA does not comprise a 2′-fluoro nucleotide.
14 . The dsRNA agent of claim 1 , wherein the sense strand contains only two 2′-deoxy modifications and the antisense strand contains only three, four, five or six 2′-deoxy modifications.
15 . The dsRNA agent of claim 1 , wherein the ligand is coupled to the 3′-end, 5′-end, and/or at an internal position of the sense strand.
16 . The dsRNA agent of claim 1 , wherein the ligand is a cell or tissue targeting agent.
17 . The dsRNA agent of claim 16 , wherein the ligand is an antibody.
18 . The dsRNA agent of claim 1 , wherein the ligand is an integrin receptor ligand.
19 . The dsRNA agent of claim 1 , wherein the ligand is a lipid or cholesterol.
20 . The dsRNA agent of claim 1 , comprising a two nucleotide overhang at the 3′-end of the antisense strand and a blunt end at the 5′-end of the antisense strand.
21 . The dsRNA agent of claim 1 , comprising blunt ends at both of the 5′-end and 3′-end of the dsRNA agent.
22 . The dsRNA agent of claim 12 , wherein the dsRNA agents have all natural nucleotides, or less than 20% non-natural nucleotides.
23 . The dsRNA agent of claim 12 , comprising a sense strand having a length of 15-17 nucleotides and an antisense strand having a length of 19-23 nucleotides.
24 . The dsRNA agent of claim 22 , comprising a two nucleotide overhang at the 3′-end of the antisense strand and a blunt end at the 5′-end of the antisense strand.
25 . The dsRNA agent of claim 22 , comprising blunt ends at both of the 5′-end and 3′-end of the dsRNA agent.
26 . The dsRNA agent of claim 12 , wherein five 2′-deoxy modifications in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand.
27 . The dsRNA agent of claim 12 , wherein at least one of the 2′-deoxy modifications is in the sense strand at position 11, counting from the 5′-end of the sense strand.
28 . The dsRNA agent of claim 12 , wherein at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.
29 . The dsRNA agent of claim 12 , wherein five of the 2′-deoxy modifications are in the antisense strand at positions 2, 5, 7, 12 and 14 counting from the 5′-end of the antisense strand, and at least two of the 2′-deoxy modifications are in the sense strand at positions 9 and 11 counting from the 5′-end of the sense strand.
30 . The dsRNA agent of claim 22 , wherein
the non-natural nucleotide is selected from the group consisting of acyclic nucleotides, locked nucleic acid (LNA) nucleotides, hexitol nucleic acid (HNA) nucleotides, cyclohexenyl nucleic acid (CeNA) nucleotides, 2′-methoxyethyl nucleotides, 2′-O-allyl nucleotides, 2′-C-allyl nucleotides, 2′-fluoro nucleotides, 2′-O—N-methylacetamido (2′-O-NMA) nucleotides, 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotides, 2′-O-aminopropyl (2′-O-AP) nucleotides, and 2′-ara-F nucleotides; and
the natural nucleotide is selected from the group consisting of 2′-OH nucleotides, 2′-OMe nucleotides, and 2′-deoxy nucleotides.
31 . The dsRNA agent of claim 12 , wherein the ligand is an ASGPR ligand.
32 . The dsRNA agent of claim 12 , wherein the dsRNA does not comprise a 2′-fluoro nucleotide.
33 . The dsRNA agent of claim 12 , wherein the sense strand contains only two 2′-deoxy modifications and the antisense strand only contains three, four, five or six 2′-deoxy modifications.
34 . The dsRNA agent of claim 12 , wherein the ligand is coupled to the 3′-end, 5′-end, and/or at an internal position of the sense strand.
35 . The dsRNA agent of claim 12 , wherein the ligand is a cell or tissue targeting agent.
36 . The dsRNA agent of claim 35 , wherein the ligand is an antibody.
37 . The dsRNA agent of claim 12 , wherein the ligand is an integrin receptor ligand.
38 . The dsRNA agent of claim 12 , wherein the ligand is a lipid or cholesterol.