In vivo knockdown, mediated by LNP01-formulated siRNAs, of HCV pro-viral factor ISG15 increased responsiveness to Interferon

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). ISG15 has been identified as pro-viral host factor promoting HCV replication. Therefore, hepatocyte-specific suppression of ISG15 expression may be a promising approach to overcome non-response to standard combination treatment of HCV patients and may lead to new therapeutic concepts in the future.

Methods: Nanolipid-formulated (LNP01) siRNAs preferentially targeting the liver were injected into C57Bl/6 mice. After different time points (6h, 24h, 48h, 72h, 96h and 10 days) tissue (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 knockdown efficiency and induction of immune responses hepatocytes and a mix of non parenchymal liver cells were isolated 6h and 48h after application of siRNAs.

Results: Application of ISG15-specific siRNA led to 80% knockdown efficiency in hepatocytes at least 10 days after injection, while no knockdown was detectable in NPCs or any other reviewed organs. 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. Intravenous administration of consensus interferon was followed by up-regulation of ISGs in liver tissue. ISG15 knockdown, induced by non-immune-activating siRNA, significantly enhanced the hepatic expression of ISGs (IFI-T1, IFI-TM3, RSAD2, MX2, Rnasel1, IFI-T3) in response to Poly I:C or interferon. However, Poly I:C had no additional stimulatory effect in combination with immune-activating siRNA.

Conclusions: A selective suppression of ISG15 expression in murine hepatocytes can be achieved, using ISG15-specific siRNAs. ISG15 suppression significantly increased the responsiveness to exogenous as well as endogenous interferon, thereby reproducing recently published data generated in vitro. This approach may lead to novel therapeutic options for the treatment of HCV patients in the future.