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861VUS and 855VUS mRNA injections rescue SMA smn−/− zebrafish disease hallmarks. (A) smn−/− zebrafish (column 3) develop morphological malformations from 3 dpf, being smaller with progressive body curvature and small eyes; for comparison, see control zebrafish (column 1). From 4 dpf, smn−/− animals develop progressive pericardial and cerebral edema. Animals died between 4 and 6 dpf, as determined by the absence of a heartbeat. mRNA from the known pathogenic SMN1 variant, SMN1 c.549del (path—negative control, column 7), as expected, failed to rescue this phenotype (did not prevent the appearance of these malformations). In contrast, positive controls: (i) ubiquitous transgenic expression of wt-SMN1 (Tg(SMN1), column 2), (ii) injected wt-SMN1 mRNA (WT, column 4), (iii) injected non-pathogenic variant c.462 A > G (non-path, column 8), and importantly (iv) injected c.861_864del SMN1 mRNA (861VUS, column 5) and injected c.855_858del SMN1 mRNA (861VUS, column 6) all rescued these morphological traits, with no distinguishable difference between the animals. These results robustly demonstrate that both the 861VUS and 855VUS produce functional SMN protein. Empty boxes indicate 100% batch mortality. Scale bar, 500 µm. (B) SMA smn−/− zebrafish present dramatic motor function loss at 5 dpf and died by 6 dpf. As previously demonstrated (Giacomotto et al, 2015), human wt-SMN1 transgenic ubiquitous expression (Tg(SMN1)) or injected wt-SMN1 mRNA (WT) rescue this motor function loss. Control smn−/−;Tg(SMN1) were heat-shocked once at 24 hpf to produce SMN. Similarly, the non-pathogenic SMN1 variant (non-path) and both the 861VUS and 855VUS efficiently restore motor function with no detectable significant difference. On the contrary, confirming the robustness of the approach, mRNA from the pathogenic SMN1 variant (path) failed to improve motor function with no difference from the negative control (smn−/−). Graphs represent comparisons of the total distance swum by each cohort of fish over 24 min. Each graph is representative of an experiment performed three times (Appendix Figs. S5–S4). Each data point represents one fish. Error bars represent the standard error of the mean. Statistical significance was evaluated using the Kruskal–Wallis test with Dunn’s correction for multiple comparisons. ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant. Exact P values are shown tabulated in Appendix Fig. S2. Source data are available online for this Figure. (C) Representative swimming tracks at 5, 6, and 7 dpf. Cross symbols indicate 100% mortality. (D) SMA smn−/− zebrafish (smn−/−, red dashed line) had a median survival of 5 days. Injected mRNA from the known pathogenic SMN1 variant (path, orange dashed line) did not have any effect on survival. In contrast, demonstrating restoration of Smn/SMN function, injected mRNA from 861VUS (dark gray dashed line) and 855VUS (light gray dashed line) extended the survival of smn−/− larvae similar to wt-mRNA (WT, green dashed line) and non-pathogenic variant (non-path, black dashed line) animals. ****P < 0.0001; ns, not significant. Statistical significance was evaluated using the log-rank (Mantel–Cox) test. Exact P values and median survival data are shown tabulated in Appendix Fig. S2. Source data are available online for this figure.
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