Dual-Plasmid Bionic Array-Directed Gene Electrotransfer in HEK293 Cells and Cochlear Mesenchymal Cells Probes Transgene Expression and Cell Fate.

Naked plasmid DNA electrotransfer offers advantages over viral-based gene delivery, including being regulatory permissive, but factors influencing expression efficiency and cell fate impact on translational utility. This study compared co-expression of red and green fluorescence reporter plasmids with differing promoters in HEK293 cells and in vivo in guinea pig cochlear mesenchymal cells using Bionic array-Directed Gene Electrotransfer (BaDGE). A functional plasmid copy number of ∼64 was established in HEK293 cells by co-transfecting with separate CMV-actin-globin (CAGp) promoter-driven mCherry and green fluorescent protein (GFP) reporters, where cell division diluted plasmids toward discrete red or green channels from 100% co-expression to 10% over 24 days (∼17 cell cycles). Cross-talk between promoters was identified by interchanging a cytomegalovirus promoter (CMVp)-driven GFP plasmid for the CAGp-GFP plasmid. Here, expression of the CMVp-GFP plasmid dominated, while a dual CAGp-based reporter plasmid cocktail showed persistent co-expression beyond 2 weeks. In contrast, in vivo, cochlear mesenchymal cells co-transduced with CAGp-mCherry and CMVp-GFP plasmids showed stable co-expression at ∼50%, while the total transfectant numbers diminished over 2 weeks. This is consistent with a lack of mitosis in the cochlear mesenchymal cells and shows that cell type is a factor in plasmid interaction.