Fig. 3

QR-421a shows a concentration-dependent increase of USH2A exon 13 skipping in WERI-Rb1 cells (A and B) WERI-Rb1 retinoblastoma cells were treated with different concentrations of QR-421a, using either (A) transfection or (B) gymnotic uptake. Untreated and control oligo-treated cells were included as negative controls. Exon-skipping level was determined by quantification of USH2A transcripts with and without exon 13 by RT-ddPCR. Treatment with QR-421a resulted in a significant concentration-dependent increase of USH2A Δexon 13 transcripts. Data are shown as mean ± SD. Two biological replicates per treatment condition. Asterisks indicate significant differences with scrambled control oligo-treated cells (∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; one-way ANOVA followed by Dunnett’s multiple comparison test). (C) Representative image of exon 11-15 RT-PCR amplicons obtained with RNA isolated from untransfected and QR-421a-transfected WERI-Rb1 cells. QR-421a is able to induce skipping of USH2A exon 13 and does not increase the formation of other alternatively spliced USH2A transcripts. Of note, USH2A Δexon 12-13 and Δexon 12-14 transcripts are already present in untreated WERI-Rb1 cells and yield out-of-frame mRNA transcripts. (D) Sanger sequencing traces of the USH2A Δexon 13 amplicon shown in (C) confirm that the sequence of exon 13 is lacking from the transcript.