Figure 2.

Intramuscular injection of repRNA/LION activates the local muscle innate immune response in mice

(A–G) C57BL/6 mice received i.m. injection of repRNA/LION and repRNA/LNP encoding SEAP (10 μg) (n = 4). Fourteen hours later, the animals were sacrificed, and total RNA was isolated from the muscle of these animals. Transcripts were analyzed by NanoString. (A) The expression of muscle genes included in the host response panel is represented by heatmaps. The color gradient of blue to orange indicates mean subtracted normalized log₂ expression values. (B and C) repRNA-induced genes are shown as volcano plots. Genes of special interest are indicated in red. Differential expression in muscle genes between repRNA/LION and naive mice, repRNA/LNP and naive mice (B), and between repRNA/LION and repRNA/LNP (C) are shown as a volcano plot. (D) PanglaoDB cell type analysis for the differentially expressed genes in the muscle of repRNA/LION vs. repRNA/LNP. (E) NanoString annotation analysis for the differentially expressed genes in the muscle of repRNA/LION vs. repRNA/LNP. (F) Normalized expression of the genes for CD40, CD80, and CD86 in the muscle of indicated mice is shown as boxplots. (G) Normalized expression of the genes for interferons in the muscle of indicated mice is shown as boxplots. (F and G) All the boxplots show min to max values. Statistical comparison of mean values among groups was performed by ordinary one-way ANOVA and Dunnett’s multiple comparison test between values in the naive group vs. values in each repRNA-delivered group. (H–J) C57BL/6 mice received i.m. injection of repRNA/LION or repRNA/LNP expressing GFP (10 μg) (n = 5). (H and I) At indicated time points, mice were sacrificed, and their muscles and dLNs were processed using a skeletal muscle dissociation kit and collagenase-based cell isolation, respectively. The frequencies of CD11b⁺ cells in the muscle and dLNs (H) and surface CD86 expression levels on the indicated cell types (I) were determined by flow cytometry. Statistical comparison of mean values among groups was performed by multiple unpaired t tests between values in the LION group vs. values in the LNP group at each time point. (J) Mice were sacrificed at day 7 after i.m. injection of repRNA/LION or repRNA/LNP expressing GFP. Their muscle tissues were isolated, sectioned, and analyzed by multiplex immunofluorescence. CD11b and CD64 were stained with antibodies against each protein. Scale bar, 200 μm. Statistically significant results are shown as asterisks: ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; ns, not significant.