Mutation-specific sbASO efficacy in RTT patient-derived human fibroblast cells. (A) Schematic representation of the experimental design, illustrating the treatment of human fibroblast cells (hFIBs) derived from RTT patients with sbASOs for biochemical analysis of nuclear fractions. (B) List of hotspot mutations that account for 70% of all RTT mutations. The mutations used in this study are highlighted in red. Our results demonstrated that sbASOs elevated MeCP2 levels in a dose-dependent and mutation-specific manner. (C–D) The optimal concentrations of each sbASO for increasing MeCP2 levels in various RTT patient-derived cell lines. (C) sbASO.miR-22 administration increased MeCP2 levels in R133C (50 nM), R306C (125 nM), and MT R294X (1.5 nM) cell lines. No significant effect was observed in T158M, WT R270X, and WT R294X. (D) sbASO.miR-132 treatment elevated MeCP2 expression across all tested cell lines, including R133C (50 nM), T158M (27.5 nM), R306C (125 nM), WT R270X (27.5 nM), WT R294X (1.5 nM), and MT R294X (27.5 nM). (E) sbASO.miR-483 administration increased MeCP2 expression in T158M (27.5 nM), WT R270X (27.5 nM), and MT R294X (27.5 nM). No significant changes were noted in R133C, R306C, and WT R294X. n = 3–5/mutation/sbASO. Statistical significance was determined using Student's t-test, with (*) P < 0.05, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001. Outliers were identified and excluded using Grubb's test at α = 0.1. Data are presented as mean ± SEM. For representative westerns and additional details on dose-response experiments and statistical analyses, please refer to Supplemental Figures S4 and S5.
