MODIFIED siRNA MOLECULES AND USES THEREOF

• US 20080249046A1
• Filed: 06/08/2007
• Published: 10/09/2008
• Est. Priority Date: 06/09/2006
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: Judge, Adam, MacLachlan, Ian
• Granted Patent: US 7,915,399 B2
• Time in Patent Office: Days
• US Class Current: 514/44
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3521 Methyl
• CPC Class Codes: C12N 2320/53 reducing unwanted side-effects

Abstract

The present invention provides chemically modified siRNA molecules and methods of using such siRNA molecules to silence target gene expression. Advantageously, the modified siRNA of the present invention is less immunostimulatory than its corresponding unmodified siRNA sequence and retains full RNAi activity against the target sequence. The present invention also provides nucleic acid-lipid particles comprising a modified siRNA, a cationic lipid, and a non-cationic lipid, which can further comprise a conjugated lipid that inhibits aggregation of particles. Methods for identifying and/or modifying an siRNA having immunostimulatory properties are also provided.

Claims

1. A chemically synthesized double stranded short interfering nucleic acid (siNA) molecule that directs cleavage of a target RNA via RNA interference (RNAi), wherein:

(a) each strand of said siNA molecule is about 18 to about 38 nucleotides in length;

(b) one strand of said siNA molecule comprises nucleotide sequence having sufficient complementarity to said target RNA for the siNA molecule to direct cleavage of the target RNA via RNA interference; and

(c) wherein one or more nucleotides of said siNA molecule are chemically modified to reduce the immunostimulatory properties of the siNA molecule to a level below that of a corresponding siNA molecule having unmodified nucleotides.

2. The siNA molecule of claim 1, wherein said siNA molecule comprises no ribonucleotides.

3. The siNA molecule of claim 1, wherein said siNA molecule comprises one or more ribonicleotides.

4. The siNA molecule of claim 1, wherein one strand of said double-stranded siNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof, and wherein a second strand of said double-stranded siNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence or a portion thereof of said target RNA.

5. The siNA molecule of claim 4, wherein each strand comprises at least about 18 nucleotides that are complementary to the nucleotides of the other strand.

6. The siNA molecule of claim 1, wherein said siNA molecule comprises an antisense region comprising a nucleotide sequence that is complementary to a nucleotide sequence of a target gene or a portion thereof, and wherein said siNA further comprises a sense region, wherein said sense region comprises a nucleotide sequence substantially similar to the nucleotide sequence of said target gene or a portion thereof.

In vivo RNA interference. Groups of 5 Balb/C mice were treated on 3 consecutive days with lipid encapsulated siRNA (native, 2′

OMe U(S), 2′

OMe or GU(AS) ApoB-1, and native or 2′

OMe U(S) Mismatch) at 5 mg/kg by standard intravenous injection via the lateral tail vein. Body weights and general observations were recorded throughout the duration of the studies. 48 h after the final siRNA treatment, mice were sacrificed. Blood was collected by cardiac puncture for serum analysis of ApoB protein and cholesterol. Livers were weighed and collected into 6 mL RNALater (Sigma) for ApoB mRNA analysis by quantigene assay (Genospectra;

Fremont, Calif.).

Serum cholesterol was measured using a commercial cholesterol detection kit according to manufacturers instructions (Thermo electron Corp;

Melbourne, Australia).

ApoB-100 was detected in serum from individual animals by sandwich ELISA using monoclonal mouse ApoB-100 capture antibody LF3 (Zlot et al., J. Lipid Res. 40;

76-84 (1999)). Bound ApoB-100 was detected with polyclonal rabbit anti-mouse ApoB (Biodesign International;

Saco, Me.) and horseradish peroxidase-conjugated goat anti-rabbit Ig'"'"'s (Jackson Immunoresearch;

West Grove, Pa.). Serum ApoB levels were determined from A₄₅₀ values using a standard curve generated with normal mouse serum to define the linear range of the ELISA and expressed as a percentage of the PBS treated control group.

The QuantiGene assay (Genospectra) was used to quantify the reduction of mouse apoB mRNA in liver tissue after siRNA treatment. Small uniform tissue samples were taken from livers that had been collected 48 h after final injection and stored in RNAlater (Sigma). Lysates were directly used for apoB and GAPDH mRNA quantification, and the ratio of apoB and GAPDH mRNA was calculated and expressed as a group average relative to the PBS control group. Specific probe sets used for detection of mRNA were designed by Genospectra to target the following regions;

for the apoB mRNA, positions 5183-5811 of accession XM_—

137955;

for GAPDH mRNA, positions 9-319 of accession NM-008084.

It is to be understood that the above description is intended to be illustrative and not restrictive. Many embodiments will be apparent to those of skill in the art upon reading the above description. The scope of the invention should, therefore, be determined not with reference to the above description, but should instead be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. The disclosures of all articles and references, including patent applications, patents, PCT publications, and Genbank Accession Nos., are incorporated herein by reference for all purposes.

7. The siNA molecule of claim 6, wherein said antisense region and said sense region comprise about 18 to about 38 nucleotides, and wherein said antisense region comprises at least about 18 nucleotides that are complementary to nucleotides of the sense region.

8. The siNA molecule of claim 1, wherein said siNA molecule comprises a sense region and an antisense region, and wherein said antisense region comprises a nucleotide sequence that is complementary to a nucleotide sequence of RNA encoded by a target gene, or a portion thereof, and said sense region comprises a nucleotide sequence that is complementary to said antisense region.

9. The siNA molecule of claim 6, wherein said siNA molecule is assembled from two separate oligonucleotide fragments wherein one fragment comprises the sense region and a second fragment comprises the antisense region of said siNA molecule.

10. The siNA molecule of claim 6, wherein said sense region is connected to the antisense region via a linker molecule.

11. The siNA molecule of claim 10, wherein said linker molecule is a polynucleotide linker.

12. The siNA molecule of claim 10, wherein said linker molecule is a non-nucleotide linker.

13. The siNA molecule of claim 6, wherein pyrimidine nucleotides in the sense region are 2′

-O-methylpyrimidine nucleotides.

14. The siNA molecule of claim 6, wherein purine nucleotides in the sense region are 2′

-deoxy purine nucleotides.

15. The siNA molecule of claim 6, wherein pyrimidine nucleotides present in the sense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides.

16. The siNA molecule of claim 9, wherein the fragment comprising said sense region includes a terminal cap moiety at a 5′

-end, a 3′

-end, or both of the 5′ and

3′

ends of the fragment comprising said sense region.

17. The siNA molecule of claim 16, wherein said terminal cap moiety is an inverted deoxy abasic moiety.

18. The siNA molecule of claim 6, wherein pyrimidine nucleotides of said antisense region are 2′

-deoxy-2′

-fluoro pyrimidine nucleotides.

19. The siNA molecule of claim 6, wherein purine nucleotides of said antisense region are 2′

-O-methyl purine nucleotides.

20. The siNA molecule of claim 6, wherein purine nucleotides present in said antisense region comprise 2′

-deoxy-purine nucleotides.

21. The siNA molecule of claim 18, wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3′

end of said antisense region.

22. The siNA molecule of claim 6, wherein said antisense region comprises a glyceryl modification at a 3′

end of said antisense region.

23. The siNA molecule of claim 9, wherein each of the two fragments of said siNA molecule comprise about 21 nucleotides.

24. The siNA molecule of claim 23, wherein about 19 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule and wherein at least two 3′

terminal nucleotides of each fragment of the siNA molecule are not base-paired to the nucleotides of the other fragment of the siNA molecule.

25. The siNA molecule of claim 24, wherein each of the two 3′

terminal nucleotides of each fragment of the siNA molecule are 2′

-deoxy-pyrimidines.

26. The siNA molecule of claim 25, wherein said 2′

-deoxy-pyrimidine is 2′

-deoxy-thymidine.

27. The siNA molecule of claim 23, wherein all of the about 21 nucleotides of each fragment of the siNA molecule are base-paired to the complementary nucleotides of the other fragment of the siNA molecule.

28. The siNA molecule of claim 23, wherein about 19 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a target gene or a portion thereof.

29. The siNA molecule of claim 23, wherein about 21 nucleotides of the antisense region are base-paired to the nucleotide sequence of the RNA encoded by a target gene or a portion thereof.

30. The siNA molecule of claim 9, wherein a 5′

-end of the fragment comprising said antisense region optionally includes a phosphate group.

31. A composition comprising the siNA molecule of claim 1 in an pharmaceutically acceptable carrier or diluent.

32. A method for generating the siNA molecule of claim 1, comprising:

(a) introducing modified nucleotides in one or more of the nucleotide positions of the siNA molecule, and

(b) assaying the siNA molecule of step (a) under conditions suitable for isolating an siNA molecule having reduced immunostimulatory properties compared to a corresponding siNA molecule having unmodified nucleotides.

33. The method of claim 32, wherein said reduced immunostimulatory properties comprise an abrogated or reduced induction of inflammatory or proinflammatory cytokines in response to the siNA being introduced in a cell, tissue, or organism.

34. The method of claim 32, wherein said reduced immunostimulatory properties comprise an abrogated or reduced induction of Toll Like Receptors (TLRs) in response to the siNA being introduced in a cell, tissue, or organism.

35. The method of claim 32, wherein said reduced immunostimulatory properties comprise an abrogated or reduced induction of interferons in response to the siNA being introduced in a cell, tissue, or organism.

36. The method of claim 33, wherein said cytokine comprises interleukin-6 (IL-6) or tumor necrosis alpha (TNF-α

).

37. The method of claim 34, wherein said Toll Like Receptor comprises TLR3, TLR7, TLR8 or TLR9.

38. The method of claim 35, wherein said interferon comprises interferon alpha.