Modified RNAi agents

Patent metadata

Field	Value
Publication number	US-9796974-B2
Application number	US-201214358009-A
Country	US
Kind code	B2
Filing date	Nov 16, 2012
Priority date	Nov 18, 2011
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

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 duplex is represented by formula (III):

sense:

5′ n p  -N a  -(X X X)  i -N b  -Y Y Y -N b  -(Z Z Z) j  -N a  -

n q  3′

antisense:

3′ n p ′-N a ′-(X′X′X′) k -N b ′-Y′Y′Y′-N b ′-(Z′Z′Z′) l -N a ′-

n q ′ 5′

(III)

wherein:
i, j, k, and l are each independently 0 or 1, provided that at least one of i, j, k, and l is 1;
p and q are each independently 0-6;
each Na and Na′ independently represents an oligonucleotide sequence comprising 2-20 nucleotides which are either modified or unmodified or combinations thereof, each sequence comprising at least two differently modified nucleotides, each Nb and Nb′ independently represents an oligonucleotide sequence comprising 1-10 modified nucleotides;
each n p , n p ′, n q and n q ′ independently represents an overhang nucleotide sequence comprising 0-6 nucleotides; and
XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides; and
wherein the modification on Nb is different than the modification on Y and the modification on Nb′ is different than the modification on Y′.

2. The double-stranded RNAi agent of claim 1 , wherein i is 1; j is 1; or both i and j are 1.

3. The double-stranded RNAi agent of claim 1 , wherein k is 1; l is 1; or both k and l are 1.

4. The double-stranded RNAi agent of claim 1 , wherein the YYY motif occurs at or near the cleavage site of the sense strand.

5. The double-stranded RNAi agent of claim 1 , wherein the Y′Y′Y′ motif occurs at the 11, 12 and 13 positions of the antisense strand from the 5′-end.

6. The double-stranded RNAi agent of claim 5 , wherein the Y′ is 2′-OMe.

7. The double-stranded RNAi agent of claim 1 , wherein formula (III) is represented as formula (IIIa):

5′ n p  -N a  -Y Y Y -N b  -Z Z Z -N a  -n q  3′

3′ n p ′-N a ′-Y′Y′Y′-N b ′-Z′Z′Z′-N a ′-n q ′ 5′

(IIIa)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

8. The double-stranded RNAi agent of claim 1 , wherein formula (III) is represented as formula (IIIb):

5′ n p -N a -X X X -N b -Y Y Y -N a -n q  3′

3′ n p -N a -X′X′X′-N b -Y′Y′Y′-N a -n q  5′

(IIIb)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides.

9. The double-stranded RNAi agent of claim 1 , wherein formula (III) is represented as formula (IIIc):

5′ n p -N a -X X X -N b -Y Y Y -N b -Z Z Z -N a -n q  3′

3′ n p -N a -X′X′X′-N b -Y′Y′Y′-N b -Z′Z′Z′-N a -n q  5′

(IIIc)

wherein each N b and N b ′ independently represents an oligonucleotide sequence comprising 1-5 modified nucleotides and each N a and N a ′ independently represents an oligonucleotide sequence comprising 2-10 modified nucleotides.

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

11. The double-stranded RNAi agent of claim 10 , wherein the duplex region is 17-19 nucleotide pairs in length.

12. The double-stranded RNAi agent of claim 10 , wherein the duplex region is 27-30 nucleotide pairs in length.

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

14. 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.

15. The double-stranded RNAi agent of claim 14 , wherein the nucleotides are modified with either 2′-OCH 3 or 2′-F.

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

17. The double-stranded RNAi agent of claim 16 , wherein the ligand is a one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

18. 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.

19. The double-stranded RNAi agent of claim 16 , wherein the ligand is attached to the 3′ end of the sense strand.

20. The double-stranded RNAi agent of claim 1 , further comprising at least one phosphorothioate or methylphosphonate internucleotide linkage.

21. The double-stranded RNAi agent of claim 1 , wherein the nucleotide at the 1 position of the 5′-end of the duplex in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT.

22. 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.

23. The double-stranded RNAi agent of claim 1 , wherein the Y nucleotides contain a

Assignees

• Alnylam Pharmaceuticals Inc

Inventors

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

Classifications

• C12N2310/343

  having patterns, e.g. ==--==--==-- · CPC title

• C12N2310/33

  of the base · 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

• A61K48/00 Primary

  Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy · CPC title

• A61P31/18

  for HIV · CPC title