Figure 3.

PE3-mediated correction of c.3631C > T (Q1211X) and c.2005C > T (R669X) COL7A1 in primary fibroblasts derived from two RDEB patients

(A and B) Schematic diagram of the pegRNA and ngRNA target sites in the COL7A1 gene in Pat1 (A) and Pat2 (B). Target sequences are highlighted in bold type, and PAM sequences are underlined. The pathogenic mutations, converted pathogenic mutations, and synonymous mutations for PAM disruption are shown in red, blue, and green, respectively (top). A 14-nt RTT and a 13-nt PBS were used for pegRNAs (bottom). (C and D) Heatmaps visualizing conversion rates determined by high-throughput sequencing. (E) Prime editing efficiencies and indel frequencies in the target sequence in the patient-derived fibroblasts transfected with various pegRNAs with different PBS lengths (n = 1–3). (F) Conversion rates in mRNA from PE-treated RDEB fibroblasts. (G) Western blot to measure C7 abundance in cell lysates and culture supernatants using GAPDH and Ponceau as internal controls, respectively. Images are representative of three independent experiments. Protein band densities from three independent experiments are presented as bar graphs. Each density value was normalized to the loading control value and expressed relative to the value in NHDFs. Data are the mean ± SEM (n = 3); ∗∗p < 0.01, ∗∗∗p < 0.001. (H) Immunofluorescence staining to visualize the C7 protein (green) in NHDFs, non-edited RDEB fibroblasts from Pat1, and PE3-treated RDEB fibroblasts. Nuclei were stained with DAPI (blue). Shown are representative images from three independent cell samples. Scale bars, 50 μm. (I) Representative graphics of flow cytometry assay for C7 restoration and quantitation of the percentage of C7-positive cells (n = 2). Histograms for unstained control and secondary antibody control for each cell samples are also shown. (J) Trypsin-based cell detachment assay. Cell adhesion is represented as the percentage of cells that remain attached after the indicated period of trypsin treatment. Five independent experiments were performed. Data are the mean ± SEM; ∗p < 0.05, ∗∗p < 0.01. (K) The proliferation of NHDFs, non-edited fibroblasts from Pat1, and PE3-treated RDEB fibroblasts was evaluated by the WST-1 assay. The ratio of the absorbance at 24, 48, and 72 h to that at 0 h is shown. Five independent experiments were performed. Data are the mean ± SEM; ∗p < 0.05, ∗∗p < 0.01. Statistical analyses were performed using two-tailed, unpaired Student’s t test for (G), (J), and (K) comparing NHDF versus Pat1 group and Pat1 group versus Pat1-peg1 group.