Fig. 6

Destabilizing seed-mediated base-pairing minimizes off-target effects and mitigates hepatotoxicity. a Bad actor siRNA-5 containing a single thermally destabilizing glycol nucleic acid (GNA) nucleotide at position seven of the antisense strand. b Volcano plots depicting global gene expression changes in rat hepatocytes at 24 h after transfection with 10 nM of parent or GNA-modified GalNAc-siRNAs. N = 3 technical replicates. c Liver exposures for parent and seed-modified siRNA-5 in rat toxicity study as assessed by stem-loop reverse transcription-quantitative PCR (RT-qPCR) for the antisense strand (AS) at necropsy (nx). d Liver RISC loading as assessed by stem-loop RT-qPCR for the antisense strand at necropsy. e Serum glutamate dehydrogenase (GLDH) 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. f H&E staining of liver sections collected at necropsy. The toxic parent siRNA-5 had fibrosis (circle), hepatocellular degeneration (bracket), single cell necrosis (*), increased mitoses (^), 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 GNA-modified siRNA-5 had degeneration, single cell necrosis, increased mitoses, and vacuolation but at a lower incidence and severity grade than the parent siRNA-5. Microscopic liver findings for all tested siRNAs are tabulated in Supplementary Table 7. N = 4 males (6–8 weeks old) per group; qw weekly dosing, GNA glycol nucleic acid