Figure 5.

Co-administration of PPMO and OEC in epithelial cells. (A, B) Efficacy of co-administration. Cells were treated with PPMO654 (1 μM) plus OEC (10 μM) by either sequential or co-administration. (A) EGFP expression quantification in HeLa EGFP654 cells incubated for 2–20 h; n = 3, *P < 0.01 versus control; 4 or 6 h co-administration and 6 or 20 h sequential are not significantly different. (B) Well-differentiated primary EGFP654 MTEC (see Methods) were imaged live by confocal microscopy (top, xy and bottom, xz confocal planes) showing that 20 h sequential and 6 h co-administration treatments were effective in eliciting correction and expression of EGFP in both ciliated epithelial cells (CC, red cilia stained with Cell Mask^®) and non-ciliated secretory cells (SC). Note: expression of EGFP at different levels in a random pattern; bar = 20 μm. (C, D) Extended splice correction effect of PPMO plus OEC in EGFP654 MTEC. Well-differentiated cells were treated once with PPMO plus OEC by co-administration for 6 h as above. (C) Fluorescence in live cultures was measured 2d, 7, 14 and 21 days post-treatment in an Azure Scanner System using identical parameters in all the scanning sessions (intensity, resolution, and height scan position) (n = 4). Note: the outer circle is the holding well (no cells) while the inner circle (6.5 mm diameter) is the permeable insert containing the live MTEC; cells were 100% confluent and well differentiated at all time points and exhibited a mosaic pattern of EGFP expression levels. (D) Parallel cultures were subjected to RNA isolation and RT-PCR analysis as before (n = 2). Co-administration of PPMO and OEC via the basolateral (BL) and apical (AP) sides was efficient and had extended effects in correcting the EGFP splicing defect in airway epithelial cell cultures.