Development of DNA delivery system using Pseudomonas exotoxin A and a DNA binding region of human DNA topoisomerase I

Gene therapy is defined as the delivery of a functional gene for expression in somatic tissues with the intent to cure a disease. Thus, highly efficie nt gene transfer is essential for gene therapy. Receptor-mediated gene deli very can offer high efficiency in gene transfer, but several technical diff iculties need to be solved. In this study, we first examined the DNA bindin g regions of the human DNA topoisomerase I (Topo I), using agarose gel mobi lity shift assay, in order to identify sites of noncovalent binding of huma n DNA Topo I to plasmid DNA. We identified four DNA binding regions in huma n DNA Topo I. They resided in aa 51-200, 271-375, 422-596. and 651-696 of t he human DNA Topo I. We then used one of the four regions as a DNA binding protein fragment in the construction of a DNA delivery vehicle. Based on th e known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo II we fused the receptor binding and membrane translocation domains of PE with a highly positively charged DNA binding region of the N- terminal 198 amino acid residues of human DNA Topo I. The resulting recombi nant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a gen eral DNA delivery vehicle without DNA sequence, topology, and cell-type spe cificity. The DNA delivery protein could be used to target genes of interes t into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy.