Figure 6.

The Efficiency of ASOs in Pseudo-Exon Skipping by Gymnotic Delivery

(A) Representative images of PCR products from fibroblasts treated with ASO-5, ASO-6, ASO-5/6, or ASO-9 in 2′-MOE backbone for 4 days without the use of any transfection reagent. (B) Quantitative real-time PCRs of total COL6A1 and mutant COL6A1 transcripts were performed in RNA samples collected from four patient skin fibroblasts treated with ASO-5, ASO-6, or ASO-5/6 at concentrations ranging from 50 to 800 nM. Data are presented as mean ± SD. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001). (C) Representative images of ECM collagen VI protein expression in patient skin fibroblasts treated with ASO-5, ASO-6, ASO-5/6, and ASO-9 at 800 nM for 4 days with gymnotic delivery. Collagen VI protein (in green) and nuclei (in blue) are displayed by immunofluorescence staining. The linear collagen VI microfibrils are indicated by white arrows and the discontinuous collagen VI microfibrils by yellow asterisks. Scale bar: 50 μm. (D) Mean intensity and the area covered by collagen VI were quantified in fibroblasts after the gymnotic delivery of ASO-5, ASO-6, ASO-5/6, and ASO-9 at 800 nM. Data represent mean ± SD. Data were analyzed by one-way ANOVA and post-Bonferroni test (∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001).