Fig. 1

Blocking RISC loading mitigates hepatotoxicity. a Structures of nucleotide analogs used at 5′-ends of siRNAs to prevent 5′-phosphorylation thus reducing RISC loading. b Liver exposures for parent (RNAi-active) and capped (RNAi-inactive) GalNAc-siRNAs in rat and mouse toxicity studies as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). Dashed vertical lines demarcate studies conducted separately. c Serum alanine aminotransferase (ALT) levels measured at necropsy. Differences between group means were evaluated for statistical significance using one way ANOVA with post hoc corrections (for multiple siRNAs) in GraphPad Prism 7. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Error bars represent standard deviation of the mean. d H&E staining of liver sections collected at necropsy. In the rat, hepatotoxic siRNAs (siRNA-1 shown here) had hepatocellular degeneration (bracketed area), increased sinusoidal cells due to Kupffer cell hyperplasia and/or leukocyte infiltration (#), single cell necrosis (*), increased mitoses (^), and hepatocellular vacuolation (arrow). In the mouse, hepatotoxic siRNAs (siRNA-7 shown here) were associated with single-cell necrosis and lower incidence and severity of the other findings commonly seen in the rat. Capped RNAi-inactive siRNAs had minimal vacuolation or no histologic findings in both species. Cytoplasmic clearing present in the mice was consistent with glycogen due to incomplete fasting and was not considered test article-related. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 1. N = 3 males (6–8 weeks old) per group; qw, weekly dosing; q2d, every other day dosing