Hepatic, MyD88-dependent toll-like receptors are activated by in vivo application of LNP01-formulated siRNAs

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 may suppress immunologically mediated off-target effects in isolated liver cells in vitro. 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 the liver, were injected into C57Bl/6, TLR3-/- and MyD88-/- mice. After different time points (6h, 24h, 48h, 72h and 96h) different tissues (liver, heart, spleen, kidney) and blood were prepared and expression of IFN-β, ISG15, IFI-T1, RSAD2, MX2, TNF-α, IL-6 and IL-10 was determined by qtRT-PCR or western blot. To analyze cell-specific induction of immune responses hepatocytes and a mix of non parenchymal liver cells (NPC) were isolated 6h and 48h after application of LNP01-formulated siRNAs.

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. LNP01-siRNAs, harbouring ribose-modified nucleotides (2'O-methyl or 2'-fluor), did not induce these off-target effects even in wild-type mice. Intravenous administration of the interferon-inducing TLR3 ligand Poly I:C led to a strong induction of IFN-β, ISG15, IFI-T1, RSAD2, MX2, TNF-α, IL-6 and IL-10, whereas in hepatocytes only ISGs were up-regulated as response to endogenous interferon. In combination with immune-activating LNP01-siRNA Poly I:C had no additional stimulatory effect.

Conclusions: These data enhance our understanding of siRNA modifications to avoid stimulatory effects upon the innate immune system of the liver via endosomal TLRs. This is of major importance for the development of safe and efficient RNAi-based therapeutics.