Figure 2
Investigation of RTM binding regions within the target intron 46/exon 47 of COL7A1 and their trans-splicing efficiencies analysed by flow cytometry. Eighty individual clones of the RTM library were analysed for the presence of a BD sequence by sequence analysis. (A) Five single RTMs, termed RTM02, RTM28, RTM37, RTM40 and RTM78, contained one or more specific BD(s) complementary to the selected COL7A1 target region intron 46/exon 47. These RTMs were included in initial experiments to investigate their potential to induce accurate trans-splicing into COL7A1. (B) Co-transfection of COL7A1-MG together with a representative low-efficiency (RTM02) (b) or high-efficiency RTM (RTM28) (c) into HEK293 cells resulted in the expression of GFP in ~40% or ~88% of all analysed cells, respectively, upon accurate 3′ RNA trans-splicing. Similar results were achieved after RTM02 (e) and RTM28 (f) transfection into the COL7A1-MG-CL-expressing cell line, in which ~40% and ~78% GFP expression, respectively, were detectable by flow cytometric analysis. GFP expression was undetectable in HEK293 cells exclusively transfected with RTM28 (a) and in the COL7A1-MG-CL-expressing cell line (d). Regardless of whether the COL7A1-MG was transiently or stably expressed in HEK293 cells, the RTM efficiencies were comparable. The most efficient RTMs, RTM28, RTM37 and RTM40, showed higher trans-splicing efficiencies than RTM02 and RTM78 in both screening settings, measured by flow cytometric analysis.
