Modified siRNA molecules and uses thereof

• US 8,188,263 B2
• Filed: 01/23/2009
• Issued: 05/29/2012
• Est. Priority Date: 11/02/2005
• Status: Active Grant

• Current Assignee: Arbutus Biopharma Corporation
• Original Assignee: Protiva Biotherapeutics Inc (Arbutus Biopharma Corporation)
• Inventors: MacLachlan, Ian, Judge, Adam, Sood, Vandana, Lee, Amy C. H.
• Primary Examiner(s): WHITEMAN, BRIAN A
• Application Number:
  US12/359,119
• Publication Number:
  US 20090270481A1
• Time in Patent Office: 1,222 Days
• Field of Search: 536/24.5
• US Class Current: 536/24.5
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 9/1272 with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids with cholesterol as the only non-phosphatidyl surfactant A61K9/127; lipids as modifying agent A61K47/543
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2320/30 Special therapeutic applications
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance

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 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. The present invention further provides methods of silencing gene expression by administering a modified siRNA to a mammalian subject. Methods for identifying and/or modifying an siRNA having immunostimulatory properties are also provided.

Claims

1. A modified siRNA comprising a double-stranded region of about 15 to about 30 nucleotides in length, wherein from 15% to about 30% of the nucleotides in the double-stranded region comprise 2′

-O-methyl (2′

OMe) nucleotides,

wherein the modified siRNA comprises 2′

OMe nucleotides in both strands of the modified siRNA,

wherein the modified siRNA comprises 2′

OMe nucleotides selected from the group consisting of 2′

OMe-guanosine nucleotides, 2′

OMe-uridine nucleotides, 2′

OMe-adenosine nucleotides, and mixtures thereof,

wherein the modified siRNA does not comprise 2′

OMe-cytosine nucleotides in the double-stranded region,

wherein the modified siRNA is less immunostimulatory than a corresponding unmodified siRNA sequence, and

wherein the modified siRNA is capable of silencing expression of a target sequence.

2. The modified siRNA of claim 1, wherein the modified siRNA comprises a double-stranded region of about 19 to about 25 nucleotides in length.

3. The modified siRNA of claim 1, wherein from about 20% to about 30% of the nucleotides in the double-stranded region comprise 2′

OMe nucleotides.

4. The modified siRNA of claim 1, wherein from about 25% to about 30% of the nucleotides in the double-stranded region comprise 2′

OMe nucleotides.

5. The modified siRNA of claim 1, wherein the modified siRNA has an interferon-α

(IFN-α

) response that is about 80% or more lower than the IFN-α

response detected for the corresponding unmodified siRNA sequence.

6. The modified siRNA of claim 1, wherein the modified siRNA comprises a 3′

overhang in both strands of the modified siRNA.

7. The modified siRNA of claim 6, wherein the 3′

overhang in one or both strands of the modified siRNA comprises unmodified nucleotides, modified nucleotides, and combinations thereof.

8. The modified siRNA of claim 1, wherein the modified siRNA comprises a sense strand sequence comprising nucleotides 1-19 of SEQ ID NO:

59.

9. The modified siRNA of claim 8, wherein the sense strand sequence comprises a 3′

overhang of two nucleotides.

10. The modified siRNA of claim 9, wherein the 3′

overhang comprises unmodified nucleotides, modified nucleotides, and combinations thereof.

11. The modified siRNA of claim 1, wherein the modified siRNA comprises a sense strand sequence consisting of SEQ ID NO:

59.

12. The modified siRNA of claim 1, wherein the modified siRNA comprises an antisense strand sequence comprising nucleotides 1-19 of SEQ ID NO:

54.

13. The modified siRNA of claim 12, wherein the antisense strand sequence comprises a 3′

overhang of two nucleotides.

14. The modified siRNA of claim 13, wherein the 3′

overhang comprises unmodified nucleotides, modified nucleotides, and combinations thereof.

15. The modified siRNA of claim 1, wherein the modified siRNA comprises an antisense strand sequence consisting of SEQ ID NO:

54.

16. The modified siRNA of claim 1, wherein the modified siRNA targets Apolipoprotein B (ApoB) and the corresponding unmodified siRNA sequence comprises the sequence:

17. A pharmaceutical composition comprising a modified siRNA of claim 1 and a pharmaceutically acceptable carrier.

18. A nucleic acid-lipid particle comprising:

(a) a modified siRNA of claim 1;

(b) a cationic lipid; and

(c) a non-cationic lipid.

19. The nucleic acid-lipid particle of claim 18, further comprising a conjugated lipid that inhibits aggregation of particles.

20. The nucleic acid-lipid particle of claim 19, wherein the conjugated lipid that inhibits aggregation of particles comprises a polyethyleneglycol (PEG)-lipid conjugate.

21. The nucleic acid-lipid particle of claim 20, wherein the PEG-lipid conjugate comprises a PEG-dialkyloxypropyl (PEG-DAA) conjugate.

22. The nucleic acid-lipid particle of claim 21, wherein the PEG-DAA conjugate is a PEG-dimyristyloxypropyl (C₁₄) conjugate.

23. The nucleic acid-lipid particle of claim 21, wherein the PEG-DAA conjugate comprises from 0.5 mol % to about 20 mol % of the total lipid present in the particle.

24. The nucleic acid-lipid particle of claim 18, wherein the cationic lipid comprises from about 2 mol % to about 60 mol % of the total lipid present in the particle.

25. The nucleic acid-lipid particle of claim 18, wherein the non-cationic lipid comprises from about 5 mol % to about 90 mol % of the total lipid present in the particle.

26. The nucleic acid-lipid particle of claim 18, wherein the non-cationic lipid comprises a phospholipid and cholesterol.

27. The nucleic acid-lipid particle of claim 18, wherein the modified siRNA is fully encapsulated in the nucleic acid-lipid particle.

28. A pharmaceutical composition comprising a nucleic acid-lipid particle of claim 18 and a pharmaceutically acceptable carrier.