The activation of the hepatic innate immune system by siRNA is controlled by chemical modifications and may involve endosomal Toll-like receptors

Abstract

Aims: siRNAs are being developed for the therapeutic use in liver cancer, viral hepatitis or metabolic disorders. Undesired immune stimulation of siRNA are usually studied in human cell lines or PBMC. Here, we assessed the capacity of chemically modified versus unmodified siRNAs to trigger immune responses in primary human cells of the liver. Methods: siRNAs targeting APOB1 (APO, APO-chol) and control siRNAs targeting luciferase (LUC) or galactosidase (GAL) mRNA were transfected into primary isolated human cells (PBMC, Kupffer cells, hepatocytes). Human hepatoma cells (Huh7) were used for comparison. Expression of IFNα, IFNβ, ISG15, IFIT1 and TNFα was determined by qt RT-PCR. Chloroquine was used as inhibitor of endosomal acidification. Results: Transfection of PBMC with APO, APO-chol and GAL siRNAs showed strong induction of IFNα/ß, ISGs and TNFα gene expression. None of these genes were induced by the LUC siRNA harbouring 2’-O-methyl modified nucleotides. Comparable results were obtained for Kupffer cells, with the cholesterol-conjugated APO siRNA giving the strongest induction in ISG expression. A more complex picture was found in hepatocytes. ISG induction was found after transfection of APO-chol siRNA and the GAL siRNA, but not for the APO and LUC siRNA. These immunestimulatory effects were abrogated after pretreatment with chloroquine. siRNA transfection of Huh7 did not lead to the induction of any ISGs despite efficient gene knockdown of APOB1 mRNA. Conclusion: Unmodified siRNA with or without cholesterol-conjugation led to strong and cell-type specific activation of the innate immune system of the liver and the peripheral blood. In contrast, 2'-O-methyl-modified siRNAs did not trigger an immune response in any tested cell type. These data further enhance our understanding of the effects of siRNA modifications on innate immune stimulation which is of major importance for the development of safe and efficient RNAi-based therapeutics.