In vivo immunostimulation. Female C57/BL6 mice were injected with LNPs containing 0.6 mg/kg siRNA or dsiRNA. The dose of dsiRNA was adjusted such that the amount of 21-mer produced by Dicer cleavage in vivo (assuming 100% accurate and complete cleavage) was equal to the amount of canonical siRNA administered. Blood was collected 4 h after injection and analyzed by Luminex assay. Twenty-three cytokines were quantified. Data were log-transformed for statistical analyses. For comparison between siRNA and dsiRNA (A), ANOVA effects of structure and sequence were used (N ≥ 2 per sequence, minimum three matched sequences). Canonical siRNAs (black circles), dsiRNAs (light gray circles), and PBS (an open square in the first column for each analyte) are depicted. (*) p < 0.05. For comparison to PBS (B), two-tailed Student's t-tests were performed. Data below LLOQ were also below PBS values and thus considered nonstimulatory. The number of compounds that stimulated at least one analyte twofold or more above PBS (p < 0.05) was tallied by group and depicted in the figure. The total numbers of stimulatory compounds are organized by analyte, with subgroups indicated by shading. Median fold stimulation over PBS (non-log values) for stimulatory compounds is indicated in each bar. Data points represent n = 3–4 and technical replicates. Modifications are (UU) unmodified/unmodified, (HL) heavy/light, and (UA) unmodified/alternating.
