MODIFIED sIRNA MOLECULES AND USES THEREOF
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.
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 - 31 . (canceled)
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.