Modified RNAi agents

Patent metadata

Field	Value
Publication number	US-12290573-B2
Application number	US-202217848850-A
Country	US
Kind code	B2
Filing date	Jun 24, 2022
Priority date	Nov 18, 2011
Publication date	May 6, 2025
Grant date	May 6, 2025

Abstract

One aspect of the present invention relates to double-stranded RNAi (dsRNA) duplex agent capable of inhibiting the expression of a target gene. The dsRNA duplex comprises one or more motifs of three identical modifications on three consecutive nucleotides in one or both strand, particularly at or near the cleavage site of the strand. Other aspects of the invention relates to pharmaceutical compositions comprising these dsRNA agents suitable for therapeutic use, and methods of inhibiting the expression of a target gene by administering these dsRNA agents, e.g., for the treatment of various disease conditions.

First claim

We claim:

1. A double-stranded RNAi agent capable of inhibiting the expression of a target gene, comprising a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, wherein the sense strand sequence is represented by formula (I):
5′ n p -N a -(XXX) i -N b -YYY-N b -(ZZZ) j -N a - n q 3′   (I)

wherein:
i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 1;
p and q are each independently 0-6;
each N a independently represents an oligonucleotide sequence comprising 2-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;
each N b independently represents an oligonucleotide sequence comprising 1, 2, 3, 4, 5, or 6 modified nucleotides;
each n p and n q independently represent an overhang nucleotide;
XXX, YYY and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides; and
YYY represents three 2′-F modified nucleotides, and the YYY motif occurs at the 9, 10 and 11 positions of the sense strand from the 5′-end;
the modification of each nucleotide next to a XXX, YYY or ZZZ motif is a different modification than the modification of the motif that the nucleotide is next to; and
at least one of the sense strand and antisense strand contains at least one phosphorothioate internucleotide modification within the first 5 nucleotides, counting from the 5′-end of the strand.

2. The double-stranded RNAi agent of claim 1 , wherein the antisense strand comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages.

3. The double-stranded RNAi agent of claim 2 , wherein the antisense strand comprises two blocks of two phosphorothioate internucleotide linkages separated by 17 phosphate internucleotide linkages.

4. The double-stranded RNAi agent of claim 1 , wherein double-stranded RNAi agent comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

5. The double-stranded RNAi agent of claim 1 , wherein double-stranded RNAi agent comprises one phosphorothioate internucleotide linkage modification within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

6. The double-stranded RNAi agent of claim 1 , wherein the duplex region is 17-30 nucleotide pairs in length.

7. The double-stranded RNAi agent of claim 6 , wherein the duplex region is 27-30 nucleotide pairs in length.

8. The double-stranded RNAi agent of claim 1 , wherein each strand has 17-30 nucleotides.

9. The double-stranded RNAi agent of claim 8 , wherein the nucleotides are modified with either 2′-OCH 3 or 2′-F.

10. The double-stranded RNAi agent of claim 1 , wherein the modifications on the nucleotides are selected from the group consisting of LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, and combinations thereof.

11. The double-stranded RNAi agent of claim 1 , wherein the modifications on the nucleotides are selected from the group consisting of 2′-O-methyl nucleotide, 2′-deoxyfluoro nucleotide, 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, 2′-ara-F, and combinations thereof.

12. The double-stranded RNAi agent of claim 1 , further comprising at least one ligand.

13. The double-stranded RNAi agent of claim 12 , wherein the ligand is one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

14. The double-stranded RNAi agent of claim 12 , wherein the ligand is attached to the 3′ end of the sense strand.

15. The double-stranded RNAi agent of claim 1 , wherein the base pair at the 1 position of the 5′-end of the duplex is an AU base pair.

16. A pharmaceutical composition comprising the double-stranded RNAi agent according to claim 1 in combination with a pharmaceutically acceptable carrier or excipient.

17. The pharmaceutical composition of claim 16 , wherein the double-stranded RNAi agent is administered through subcutaneous or intravenous administration.

18. A method for inhibiting the expression of a target gene in a subject comprising the step of administering the double-stranded RNAi agent according to claim 1 to the subject, in an amount sufficient to inhibit expression of the target gene.

19. The method of claim 18 , wherein the double-stranded RNAi agent is administered through subcutaneous or intravenous administration.

20. A method for delivering a polynucleotide to a specific target of a subject, the method comprising: delivering the double-stranded RNAi agent according to claim 1 by subcutaneous administration into the subject.

Assignees

• Alnylam Pharmaceuticals Inc

Inventors

• Rajeev Kallanthottathil G
• Zimmermann Tracy
• Manoharan Muthiah
• Maier Martin
• Kuchimanchi Satyanarayana
• Charisse Klaus

Classifications

• C12N15/85

  for animal cells · CPC title

• C07H21/02

  with ribosyl as saccharide radical · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2320/51

  modulating the chemical stability, e.g. nuclease-resistance · CPC title

• C12N2310/322

  2'-R Modification · CPC title