Fig 2: Differential protein expression between CTRL and FRDA samples.A, heat map illustrating expression levels of 30 proteins identified as significantly different (p < 0.05) between FRDA and CTRL groups. Shades of blue indicate lower expression, whereas shades of red indicate increased protein levels. B, a summary of Log2 FC expression ratios along with p values is provided. Proteins are listed in descending order based on expression change; shades of red indicate increased expression in FRDA, and shades of blue indicate reduced expression in FRDA. C, Pearson correlation coefficients were calculated for significantly changed proteins using their RPPA expression values in FRDA samples. Proteins and their correlation coefficients were organized by hierarchical clustering and are shown by the heat map (correlation matrix). The color key indicates shades of red for positive correlation coefficients (r > 0) and shades of blue for negative correlation coefficients (r < 0). CTRL, control; FC, fold change; FRDA, Friedreich's ataxia; RPPA, reverse phase protein array.

Fig 3: The RVD Sequences of the plTALEsTA and Their Target Sequences(A) Scheme representing the different molecular parts of the pCR3.1-plTALEVP64 (the CMV promoter, N-ter, repeat domain, C-ter, NLS, and VP64) constructs. Primers P7p, TALE-F, TALE-R, and BGH were used for sequencing. (B) List of plTALEVP64, plTALEp300, plTALEST10X, and plTALEST24X names with their RVD sequences, which permit binding to the targeted nucleotides. (C) Schematic localization regions 1, 2, 3, 6, 7, 8, 10, 11, F2, F3, and F4 in the promoter and in intron 1 of the FXN gene targeted by the plTALEs.

Fig 4: In vitro characterization of FXN silencing in HAs. A–C HAs were transduced with either the LV-scrambled or LV-shRNA37 for the indicated times. A Representative immunoblot and quantification of FXN levels over time after transduction. Equal amounts of protein were immunoblotted and probed with antibodies against FXN and ß-actin (as loading control) (n = 5). B Cellular metabolic activity (MTS reduction) in HAs 48, 72 and 96 h post-transduction (n = 7). C Representative immunoblot and quantification of C3 levels over time after transduction. Equal amounts of protein were immunoblotted and probed with antibodies against C3 and ß-actin (as loading control) (n = 6). HAs treated for 24 h with IL-1a, TNF-a and C1q were used as a positive control. All values were normalized to the untreated group (data not plotted) of their corresponding time point. Data in all graphs represent mean values ± S.E.M. In A–C data were evaluated by one-way ANOVA followed by Tukey’s post hoc test. In C data from the IL-1a, TNF-a and C1q treated group were compared to the untreated group (data not plotted) using a one-sample t-test

Fig 5: Mechanism of FXN activation by repeat-targeted duplex RNAs.(a) RIP examining the association of Ago2 with FXN pre-mRNA after treatment with 50 nM duplex RNA and analysis by real-time PCR. An arrow marks the PCR product of FXN pre-mRNA, which was confirmed by sequencing (Supplementary Fig. 7) (b) Anti-GAA duplex RNA with central mismatches (siGAA 9,10 mm with mismatches on both strands; 25 nM) activates FXN expression at a level similar to the analogous fully complementary duplex RNA (n=3). siExon3 is a positive control for transfection efficiency targeting exon 3 of FXN. (c) ChIP for RNAP2 using four different primer sets (n=4). (d) ChIP for transcription-associated histone modification markers H3K4me3, H3K9me2, H3K9me3, H3K9Ac, H3K27me3 and H4Ac (n=4–8). (e) FXN mRNA stability assay. Cells were transfected with duplex RNAs siGAA or CM at 25 nM (n=3). Actinomycin D (5 µg ml-1) was added with fresh media 3 days after transfection and cells were collected at the indicated time points. HPRT expression was measured for normalization. All experiments were performed in GM03816 patient-derived cells. All data are presented as mean±STDEV. *P<0.05, **P<0.01, by Student t-test.