In vivo treatment with nanolipid-formulated siRNAs targeting ISG15 leads to enhanced responsiveness to interferon in primary hepatocytes

Introduction: Non-response of HCV patients to combination therapy has previously been associated with a strong up-regulation of the interferon stimulated gene 15 (ISG15). The ISG15 gene product is a pro-viral factor for HCV replication in hepatocytes, but not in non-parenchymal liver cells (NPC). RNAi-based suppression of ISG15 expression in hepatocytes may overcome non-response to standard combination treatment. Methods: Different Nanolipid-formulated (LNP01) siRNAs, preferentially targeting hepatocytes, were injected into C57Bl/6 mice. After 6h, 48h and 10d 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. Primary hepatocytes and a mix of NPC were isolated to analyze cell-specific knockdown and immune response. Results: LNP01-ISG15 led to a sustained suppression of ISG15 expression in hepatocytes for at least 10 days after injection by about 80% while no significant knockdown was detectable in NPCs or any other tissue studied. Intravenous administration of PolyI: C led to strong induction of IFN-β, ISG15, IFI-T1, CIG5, MX2, TNF-α, IL–6 and IL–10 in liver tissue, whereas in hepatocytes only ISGs were up-regulated, as response to endogenous interferons produced by the NPCs. Intravenous administration of IFN-α led to induction of ISGs (ISG15, IFI-T1, CIG5) in liver tissue. ISG15 knockdown by LNP01-siRNA prior to systemic application of PolyI: C or IFN-α further enhanced the expression of ISGs (IFI-T1, IFI-TM3, CIG5, MX2, Rnasel1, IFI-T3) in the liver. Conclusions: The data suggest that ISG15 expression in murine hepatocytes can be selectively suppressed using LNP01 formulated ISG15-specific siRNAs. ISG15 suppression further increased the response to endogenous as well as exogenous IFNs, thus replicating our previous in vitro data. This approach may lead to novel therapeutic options for the treatment of HCV patients non-responding to IFN-based therapies.

HCV - ISG15 - interferon - primary hepatocytes - siRNA