| The development of nonviral vectors for safe and effective gene delivery system is acquiring significant consideration at the present time. Ideally a nonviral vector must overcome the barrier of extracellular milieu, cellular uptake by endocytosis, and escape from the endosomal compartments prior to trafficking to lysosomes, cytosolic transport, and nuclear localization of the plasmid. Bactofection is a technique wherein bacterial-maintained plasmid DNA is directly transferred and expressed in target mammalian cells. Despite extensive attempts to optimize the vector, lingering challenges related to cytotoxicity and immunogenicity has limited clinical relevance. To addresses these concerns, we developed the two approaches to attenuate the bacteria prior to its delivery into the cell. First we investigated the use of a polymyxin B (PLB) pre-treatment upon listeriolysin O (LLO) producing Escherichia coli BL21 (DE3), and evaluated these as a gene delivery vector to a murine RAW264.7 macrophage cell line using a 96-well high-throughput assay. Use of polymyxin B pre-treated delivery vectors resulted in statistically higher levels of gene delivery and reduced cytotoxicity, which can be attributed to PLB's ability to bind and inactivate endotoxin. Secondly we studied the effect of Lysis gene E (LyE) from bacteriophage phi X174, which resulted in considerable improvements in the gene delivery transfection efficiency and decreased cytotoxicity. This approach represents a facile way to elevate potency of bacterial-based gene delivery vectors while also limiting potential cytotoxic side-effects. Key words: Escherichia coli, gene delivery, bactofection, PLB, Lysis gene E. |
