Hepatic toll-like receptors can be activated by application of siRNAs in vivo via MyD88-dependent pathways which can be controlled by chemical ribose modifications of the siRNA backbone

Introduction: siRNAs are being developed for the therapeutic use in liver cancer, viral hepatitis, metabolic disorders and others. We have previously shown that chemical modifications of siRNAs suppress immunologically mediated off-target effects in vitro and in vivo. Here, we assessed efficacy and the Toll-like receptor dependent immune-stimulatory effects of differently modified siRNAs in vivo. Methods: Nanolipid-formulated (LNP01) siRNAs preferentially targeting hepatocytes were injected into C57Bl/6, TLR3-/- and MyD88-/- mice. After 6h and 48h tissue (liver, heart, spleen, kidney) and blood were prepared and expression of IFN-β, ISG15, IFI-T1, CIG5, MX2, TNF-α, IL–6 and IL–10 was determined by qtRT-PCR. Furthermore, hepatocytes and a mix of non-parenchymal liver cells (NPC) were isolated to analyze cell-specific knockdown efficiency and induction of immune responses. Results: LNP01 formulated, unmodified siRNA caused enhanced transient expression of ISGs and inflammatory genes (IFN-β, ISG15, IFI-T1, TNF-α, IL–6, IL–10) in the liver of wild-type and TLR3-/- mice 6h after application. In the spleen similar results were obtained although the level of gene expression was reduced, whereas in heart, kidney and blood only marginal induction of ISGs was observed. In contrast MyD88-/- mice, showed no induction of these genes in response to siRNA application. The siRNA-induced immune response in the liver was mediated by the NPCs, whereas hepatocytes only showed elevated expression of ISGs. LNP01-siRNAs, harbouring ribose-modified nucleotides (2’O-methyl or 2’fluor), did not induce these off-target effects even in wild-type mice. Conclusions: These data enhance our understanding of siRNA modifications to avoid stimulatory effects upon the innate immune system of the liver via endosomal TLRs and MyD88-dependent signalling pathways. This is of major importance for the development of safe and efficient RNAi-based therapeutics for the treatment of liver diseases.

MYD88 - TLR - immune response - siRNA