Supplementary Figure 14: Immunosuppression by FK506 administration reduces the host immune response.

(a) Cellular damage that causes myofiber degeneration and repair typically results in centrally nucleated myofibers under histological examination. Part of a histological section is shown to depict the quantification method. Delivery of minicircle-Cas9^FL or pCAG-GFP via DNA electroporation induced an increase in the fraction of centrally nucleated myofibers, compared to controls electroporated with vehicle only (n = 4 mice per condition) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; one-way ANOVA, followed by Tukey-Kramer test). FK506 reduced but did not fully mitigate the elevated fraction of centrally nucleated myofibers. (b) FK506 reduces CD45+ immune cell infiltration in muscles electroporated with minicircle-Cas9^FL and/or pCAG-GFP as assessed by immunofluorescence. Gray lines, histograms of CD45 fluorescence intensity around each myofiber per muscle histological section; black solid lines, mean distributions of histograms (n = 4 mice per condition, 2 sections per mouse). (c) FK506 reduces immune cell infiltration in transgene-expressing muscles as assessed by FACS (n = 4 mice per condition) (*, P < 0.05; **, P < 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against uninjected muscles). Checkmarks denote injected vectors and conditions. (d) FK506 reduces the elevated intramuscular IgG and IgM antibody levels induced by electroporation of vectors expressing Cas9 and/or GFP (n = 4 mice per condition). Scale bar, 200 μm. (e) FK506-treated mice show significantly lower body weights compared to vehicle-injected mice, manifesting signs of expected adverse reactions towards broad-spectrum immunosuppression (n = 3 mice per condition) (one-tailed Welch’s t-test, assuming unequal variances). Red lines, means.