Exploiting the virulence factor LLO for enhanced non-viral DNA delivery through forced lysosomal escape

The introduction of therapeutic genes into cells by non-viral gene delivery systems is still limited by low transfection efficacy. The degradation of the introduced DNA inside the lysosome after uptake is one of the main limiting factors of non-viral gene delivery.The aim of this study was to exploit natural endosomal escape mechanisms to overcome this transfection barrier. Listeriolysin O (LLO), a pH dependent hemolytic and endosomolytic protein derived from Listeria monocytogenes was expressed in E. coli and purified by Ni-NTA chromatography. The pH restricted hemolytic activity of the purified protein in acidic (lysosomal) pH conditions (active only at pH < 6) was confirmed by lysis of red blood cells.In following in vitro experiments, C2C12 cells were transfected with fluorophore labeled DNA with or without LLO via poly-L-lysine. Using live confocal laser scanning microscopy, a substantial decrease of lysosomal trafficking of labeled DNA was observed compared to controls without LLO within 20min after transfection. The described results demonstrate the possibility of partially adapting the entry mechanism of L. monocytogenes to increase DNA release to the cytosol by disrupting the acidifying late endosomes of the target cells and hence potentially increase the efficiency of gene delivery. Additions of electrostatic or sequence-specific DNA binding motifs to LLO resulting in a more efficient co-delivery are currently under investigation. This project was partially funded by EXPERTISSUES.