RESUMO
Bacteria-mediated gene transfer ('bactofection') has emerged as an alternative approach for genetic vaccination and gene therapy. Here, we assessed bactofection of airway epithelial cells in vitro and in vivo using an attenuated Escherichia coli genetically engineered to invade non-phagocytic cells. Invasive E. coli expressing green fluorescent protein (GFP) under the control of a prokaryotic promoter was efficiently taken up into the cytoplasm of cystic fibrosis tracheal epithelial (CFTE29o-) cells and led to dose-related reporter gene expression. In vivo experiments showed that following nasal instillation the vast majority of GFP-positive bacteria pooled in the alveoli. Further, bactofection was assessed in vivo. Mice receiving 5 x 10(8) E. coli carrying pCIKLux, in which luciferase (lux) expression is under control of the eukaryotic cytomegalovirus (CMV) promoter, showed a significant increase (P<0.01) in lux activity in lung homogenates compared to untransfected mice. Surprisingly, similar level of lux activity was observed for the non-invasive control strain indicating that the eukaryotic CMV promoter might be active in E. coli. Insertion of prokaryotic transcription termination sequences into pCIKLux significantly reduced prokaryotic expression from the CMV promoter allowing bactofection to be detected in vitro and in vivo. However, bacteria-mediated gene transfer leads to a significantly lower lux expression than cationic lipid GL67-mediated gene transfer. In conclusion, although proof-of-principle for lung bactofection has been demonstrated, levels were low and further modification to the bacterial vector, vector administration and the plasmids will be required.