Chirally-enriched double-stranded RNA agents
The present invention relates to a chirally-modified dsRNA agent capable of inhibiting the expression of a target gene. The sense and antisense strands of chirally-modified dsRNA agent independently or in combination comprises one or more site specific-site specific/position specific, chirally-modified internucleotide linkages.
1. A chirally-modified double-stranded RNA (dsRNA) agent capable of inhibiting the expression of a target gene, said chirally-modified dsRNA agent comprising a sense strand and an antisense strand, each strand having 14 to 40 nucleotides,
wherein:
the sense strand comprises one or more terminal, chirally-modified internucleotide linkages at the 5′ end;
the antisense strand comprises one or more terminal, chirally-modified internucleotide linkages at the 5′ end and one or more terminal, chirally-modified internucleotide linkages at the 3′ end;
each terminal, chirally-modified internucleotide linkage is a phosphorothioate linkage;
a terminal, chiral modification occurs at the first internucleotide linkage at the 3′ end of the antisense strand, and the linkage phosphorus atom is in Sp configuration; and
a terminal, chiral modification occurs at the first internucleotide linkage at the 5′ end of the antisense strand, and the linkage phosphorus atom is in Rp configuration.
2. The chirally-modified dsRNA agent of claim 1 , wherein the chiral purity with respect to the chiral linkage phosphorus atom for each terminal, chirally-modified internucleotide linkage is at least 50%.
3. The chirally-modified dsRNA agent of claim 1 , wherein a terminal, chiral modification occurs at the first internucleotide linkage at the 5′ end of the sense strand, and the linkage phosphorus atom is in either Rp configuration or Sp configuration.
4. The chirally-modified dsRNA agent of claim 1 , wherein the chirally-modified dsRNA agent comprises:
a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.
5. The chirally-modified dsRNA agent of claim 1 , wherein the chirally-modified dsRNA agent comprises four or more terminal, chirally-modified internucleotide linkages.
6. The chirally-modified dsRNA agent of claim 5 , wherein the chirally-modified dsRNA agent comprises:
a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
7. The chirally-modified dsRNA agent of claim 1 , wherein the chirally-modified dsRNA agent comprises five or more terminal, chirally-modified internucleotide linkages.
8. The chirally-modified dsRNA agent of claim 7 , wherein the chirally-modified dsRNA agent comprises:
a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
9. The chirally-modified dsRNA agent of claim 7 , wherein the chirally-modified dsRNA agent comprises:
a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration,
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
10. The chirally-modified dsRNA agent of claim 7 , wherein the chirally-modified dsRNA agent comprises:
a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration,
a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and
a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.
11. The chirally-modified dsRNA agent of claim 1 , wherein the chirally-modified dsRNA agent comprises six or more terminal, chirally-modified internucleotide linkages.
12. The chirally-modified dsRNA agent of claim 1 , wherein the chirally-modified dsRNA agent comprises eight or more terminal, chirally-modified internucleotide linkages.
13. The chirally-modified dsRNA agent of claim 1 , wherein each nucleotide of a dinucleotide connected by the terminal, chirally-modified internucleotide linkage is independently modified with a modification selected from the group consisting of acyclic nucleotides, LNA, HNA, CeNA, 2′-O-methoxyalkyl, 2′ O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O-N-methylacetamido (2′ O NMA), 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′-O-aminopropyl (2′ O AP), and 2′-ara F.
14. The chirally-modified dsRNA agent of claim 1 , wherein each of the sense and antisense strands is independently modified with a modification selected from the group consisting of acyclic nucleotides, LNA, HNA, CeNA, 2′ O-methoxyalkyl, 2′ O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-fluoro, 2′-O-N-methylacetamido (2′-O-NMA), 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE), 2′ O aminopropyl (2′-O-AP), and 2′-ara-F.
15. The chirally-modified dsRNA agent of claim 1 , wherein each of the sense and antisense strands has 15-30 nucleotides.
16. The chirally-modified dsRNA agent of claim 15 , wherein the sense strand has 19-22 nucleotides, and the antisense strand has 19-25 nucleotides.
17. The chirally-modified dsRNA agent of claim 1 , further comprising at least one ASGPR ligand.
18. The chirally-modified dsRNA agent of claim 17 , wherein the ASGPR ligand is attached to the 3′ end of the sense strand or antisense strand.
19. The chirally-modified dsRNA agent of claim 17 , wherein the ASGPR ligand is attached through one or more cleavable linkers.
20. The chirally-modified dsRNA agent of claim 17 , wherein the ASGPR ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.
21. The chirally-modified dsRNA agent of claim 20 , wherein the ASGPR ligand is:
22. A pharmaceutical composition comprising the chirally-modified dsRNA agent of claim 1 and a pharmaceutically acceptable carrier or excipient.
23. A method for inhibiting the expression of a target gene comprising the step of administering the chirally-modified dsRNA agent of claim 1 in an amount sufficient to inhibit expression of the target gene.
24. The method of claim 23 , wherein the chirally-modified dsRNA agent is administered through subcutaneous or intravenous administration.