Fig. 4

QR-421a treatment induces a concentration-dependent increase of USH2A exon 13 skipping in iPSC-derived photoreceptor progenitor cells (PPCs) from a patient (USH2A^{c.2299delG/c.2299delG}) (A) Gene-expression analysis indicates successful differentiation toward PPCs. The decrease in NANOG expression is indicative for loss of pluripotency, whereas the increased expression of photoreceptor markers CRX, NRL, OPN1SW, OPN1LW, and RHO is indicative of the successful differentiation toward photoreceptor cells. (B) RT-PCR analysis of USH2A exons 11 to 15 in untreated PPCs of a patient homozygous for the c.2299delG mutation only revealed an amplicon containing exons 11 to 15. Continuous treatment of PPCs with QR-421a (28 days) specifically induced the skipping of USH2A exon 13 from the transcript. There was no evidence of alternative splice site activation in exon 13 or skipping of multiple exons. (C) Quantitative analysis of USH2A exon 13 skipping by RT-ddPCR upon continuous treatment with QR-421a. Treatment was started after 3 months of differentiation and lasted for 28 days. One-half of the culture medium was refreshed every other day with medium containing QR-421a. Skipping of exon 13 was already observed at the lowest concentration and further increased with increasing concentrations. Asterisks indicate significant differences with scrambled control oligo-treated cells (∗p < 0.05; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; mean ± SD of 3 samples per condition, one-way ANOVA followed by Dunnett’s multiple comparison test).