FEATURED ARTICLE Chemical modiications on siRNAs avoid Toll-like- receptor-mediated activation of the hepatic immune system in vivo and in vitro Ruth Broering 1 *, Catherine I. Real 1 *, Matthias J. John 2 , Kerstin Jahn-Hofmann 2 , Ludger M. Ickenstein 2 , Kathrin Kleinehr 1 , Andreas Paul 3 , Kathrin Gibbert 4 , Ulf Dittmer 4 , Guido Gerken 1 and Joerg F. Schlaak 1 1 Department of Gastroenterology and Hepatology, University Hospital of Essen, Hufelandstrasse 55, Essen, North Rhine– Westphalia 45122, Germany 2 Roche Kulmbach GmbH, Fritz-Hornschuch-Strasse 9, Kulmbach, Bavaria 95326, Germany 3 Department of General, Visceral and Transplantation Surgery and 4 Institute of Virology, University Hospital of Essen, Hufelandstrasse 55, Essen, North Rhine–Westphalia 45122, Germany Correspondence to: J. F. Schlaak; E-mail: joerg.schlaak@uni-due.de *Both authors contributed equally to this work. Received 24 October 2012, accepted 11 July 2013 Objectives: The therapeutic application of small interfering RNAs (siRNAs) is limited by the induction of severe off- target effects, especially in the liver. Therefore, we assessed the potential of differently modiied siRNAs to induce the hepatic innate immune system in vitro and in vivo. Methods: Primary isolated liver cells were transfected with siRNAs against apolipoprotein B1 (APOB1), luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in lipid nanoparticles (LNPs) and administered intravenously to C57BL/6 mice. Liver tissue was collected 6–48 h after injection and knock-down efficiency or immune responses were determined by quantitative reverse-transcription-linked PCR. Results: Unmodiied GAL siRNA transiently induced the expression of TNF-α, IL-6, IL-10, IFN-β and IFN-sensitive gene 15 in vivo, whereas a formulation of 2′-O-methylated-LUC siRNA had no such effects. Formulation of unmodiied APOB1- speciic siRNA suppressed APOB1 mRNA levels by ~80% in the liver 48 h after application. The results were paralleled in vitro, where transfection of liver cells with unmodiied siRNAs, but not with chemically modiied siRNAs, led to cell- type-speciic induction of immune genes. These immune responses were not observed in MYD88-deicient mice or in chloroquine-treated cells in vitro. Conclusions: Our data indicate that siRNAs activate endosomal Toll-like receptors in different liver-derived cell types to various degrees, in vitro. LNP-formulated siRNA selectively leads to hepatic knock-down of target genes in vivo. Here, off-target immune responses are restricted to non-parenchymal liver cells. However, 2′-O-methyl modiications of siRNA largely avoid immune-stimulatory effects, which is a crucial prerequisite for the development of safe and efficient RNA-interference-based therapeutics. Keywords: hepatocytes, innate immunity, lipid nanoparticles, non-parenchymal liver cells, siRNA Introduction The phenomenon of RNA interference (RNAi) was irst dis- covered by Fire et al. and describes post-transcriptional gene silencing of cognate target sequences, mediated by double-stranded (ds) RNAs (1). Chemically synthesized small interfering RNAs (siRNAs) of 19–24 nucleotides in length provide the means to bypass endogenous RNA- processing steps and thus to induce RNAi in eukaryotic cells (2, 3). Meanwhile, as RNAi is widely accepted as a promising tool for the development of new therapeutic concepts, piloting studies have already been performed in clinical trials (4, 5). Still, the safe and eficient delivery of RNAi-based therapeutics remains an important challenge for their clinical development. Because siRNA is highly negatively charged, the molecule cannot permeate the cell wall. Potential siRNA- based therapeutic drugs against liver diseases such as viral hepatitis thus require a delivery system for improved cellular uptake. siRNAs modiied by lipophilic moieties enhance the uptake of siRNA via a receptor-mediated mechanism or by an increased membrane permeability (6). By using lipid nanoparticle (LNP) formulations, the siRNA doses necessary to achieve suficient target knock-down in © The Japanese Society for Immunology. 2013. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com International Immunology, Vol. 26, No. 1, pp. 35–46 doi:10.1093/intimm/dxt040 Advance Access publication 24 September 2013 by guest on May 22, 2016 http://intimm.oxfordjournals.org/ Downloaded from