Fig. 4

Swapping seed regions mitigates hepatotoxicity. a Chemical structures of seed swapping between a hepatotoxic and a non-hepatotoxic GalNAc-siRNA. b Liver exposures for parent and seed-swapped GalNAc-siRNAs in rat toxicity study as assessed by stem-loop RT-qPCR for the antisense strand (AS) at necropsy (nx). c Liver RISC loading as assessed by stem-loop RT-qPCR for the antisense strand at necropsy. d 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 < 0.01, ***p < 0.001. Error bars represent standard deviation of the mean. e H&E staining of liver sections collected at necropsy. The toxic siRNA had hepatocellular degeneration (bracket), single cell necrosis (*), increased sinusoidal cells consistent with Kupffer cell hyperplasia and/or infiltrating leukocytes (#), and hepatocellular vacuolation (arrow), while the non-toxic siRNA had only minimal vacuolation. The non-toxic seed in the toxic backbone was comparable to the full non-toxic siRNA, and the toxic seed in the non-toxic backbone had single cell necrosis, increased sinusoidal cells and vacuolation but at a lower severity grade than the full-length toxic compound. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 6. N = 3 males (6–8 weeks old) per group; q2d, every other day dosing