RNA interference: Induction of the IFN Response

RNA interference (RNAi) has become a powerful tool to uncover gene function and is evolving as a new therapeutic modality. The mediators of RNAi are double stranded (ds) small interfering RNAs (siRNAs) cellularly delivered as synthetic duplexes or as short hairpin RNAs (shRNAs) by plasmids or viral vectors. siRNAs are incorporated into the RNA interference specificity complex (RISC) leading to the cleavage and degradation of the target mRNA.

A growing concern when inducing RNAi whether using synthetic siRNAs or shRNAs is the activation of an immune response. Recent studies report that siRNAs can be potent inducers of interferons (IFNs) and inflammatory cytokines both in vivo and in vitro ^[1-3] raising questions about the specificity of RNAi. Much of the IFN response is caused by the activation of the dsRNA-dependent protein kinase R (PKR) leading to a global inhibition of protein synthesis. Yet siRNAs that are shorter than 30 bp can evade PKR activation. Toll-like receptor (TLR) 3 is another receptor for dsRNA and seems to be involved in siRNA-induced IFN response^[4]. However, this recognition appears to be cell-specific as not all immune cells express TLR3. Activation of immune cells by siRNAs is also sequence dependent and sense or antisense strands separately can induce cytokine production as efficiently as ds siRNAs^[1-3]. Thus, other TLRs may play a role in the stimulatory effect of siRNAs. Recent data indicate that TLR7 and/or TLR8 mediate the recognition of ds and single strand (ss) siRNAs in a sequence-dependent manner^[1-3]. U-rich and GU-rich siRNAs seem to be preferentially recognized but some siRNAs were found to be stimulatory independently of their GU content suggesting the existence of specific sequences recognized by TLR7/8. TLR7 and TLR8 are endosomal receptors and require the compartmentalization of siRNAs in endosomes to be activated. Indeed, lipid-delivered siRNAs that localize in the endosome can be immunostimulatory in contrast to siRNAs delivered in the cytoplasm by electroporation^[3].

The immunostimulatory "side effects" of siRNAs must be taken in account when inducing RNAi. InvivoGen is introducing a set of primers designed to detect an IFN-response by real-time quantitative PCR. In order to limit this IFN response and other stimulatory effects, InvivoGen is updating the siRNA Wizard, its free siRNA-design software, to exclude sequences that are potentially immunostimulatory (sirnawizard.com). Furthermore, as shRNAs appear to be less stimulatory than synthetic siRNAs, the use of pCpG-siRNA, a CpG-free plasmid designed for the production of shRNAs that does not activate TLR9, may further limit the induction of an siRNA-induced immune response.

References:

1. Hornung V. et al., 2005. Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7. Nat Med. 11(3):263-70.
2. Judge AD. et al., 2005. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 23(4):457-62.
3. Sioud M., 2005. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence-dependent and requires endosomal localization. J Mol Biol. 348(5):1079-90.
4. Kariko K. et al., 2004. Exogenous siRNA mediates sequence-independent gene suppression by signaling through toll-like receptor 3. Cells Tissues Organs. 177(3):132-8.