The non-specific activation of liver tissue by siRNAs in vivo can be controlled by chemical ribose modifications of siRNAs

Introduction: siRNAs have been developed for therapeutic use in liver cancer, viral hepatitis or metabolic disorders. We previously showed that chemical modification of siRNAs control immunologically mediated off-target effects in isolated liver cells in vitro. In the present study, we assessed efficacy and immune-stimulatory effects of these siRNAs in vivo. Methods: Human and murine hepatocytes and non-parenchymal liver cells were transfected with siRNAs against APOB1 (APO), interferon-sensitive gene15 (ISG15) or control siRNAs against luciferase (LUC) or galactosidase (GAL). For in vivo use, siRNAs were formulated in hepatocyte-targeting lipid nanoparticles (LNP01) and injected i.v. into C57/Bl6 mice. Liver, heart, spleen, kidney, and blood samples were collected after 6–48h and mRNA levels of APOB1, IFNα, IFNβ, ISG15, IFIT1, TNFα, IL6, IL10, and TGFβ were determined by qtRT-PCR. Results: Transfection of primary liver cells with unmodified siRNAs led to cell type specific elevation of mRNAs (IFNα/ß, TNFα and ISGs). In contrast chemically modified siRNA did not induce any cytokines. The in vivo experiments revealed that administration of APO-LNP01 or ISG15-LNP01 led to 80% or 40% suppression of APOB1 or ISG15 mRNA, respectively, in the liver 48h after injection. This suggests that ISG15 expression is unaltered in non-parenchymal liver cells. In vivo administration of GAL-LNP01 resulted in expression of interferon-sensitive mRNAs transiently 6h after administration in the liver and to a lesser degree in the spleen. mRNA levels returned to basal levels 48h post-injection. Only a marginal induction of ISGs was observed in heart, kidney, and blood. In contrast, administration of (2′-O-methyl-modified) LUC-LNP01 did not induce any of these immune-stimulatory effects in vivo. Conclusion: Our study revealed that 2′-O-methyl siRNA modifications can evade immune-stimulatory effects, which is crucial for the development of safe and efficient therapeutics for RNA interference.

chmical modification - lipid nanoparticles - liver-targeting - siRNA