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

Abstract

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 efficient gene transfer is essential for gene therapy. Receptor-mediated gene delivery can offer high efficiency in gene transfer, but several technical difficulties need to be solved. In this study, we first examined the DNA binding regions of the human DNA topoisomerase I (Topo I), using agarose gel mobility shift assay, in order to identify sites of noncovalent binding of human DNA Topo I to plasmid DNA. We identified four DNA binding regions in human DNA Topo I. They resided in aa 51-200, 271-375, 422-596, and 651-696 of the 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 the known functional property of each Pseudomonas exotoxin A (PE) domain and human DNA Topo I, 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 recombinant protein was examined for DNA binding in vitro and transfer efficiency in cultured cells. The results show that this DNA delivery protein is a general DNA delivery vehicle without DNA sequence, topology, and cell-type specificity. The DNA delivery protein could be used to target genes of interest into cells for genetic and biochemical studies. Therefore, this technique can potentially be applied to cancer gene therapy.

SV40 Pseudovirion gene delivery of a toxin to treat human adenocarcinomas in mice

“…IL-4 fused with Pseudomonas exotoxin was delivered to brain tumors and destroyed the brain tumor selectively. 31,32 Chen et al 33 attempted to introduce the membrane translocation domain of Pseudomonas exotoxin A into a DNA delivery vehicle to increase the translocation efficiency of DNA into the cytosol. 34 That work, however, was cell-type dependent.…”

HER2-targeting recombinant protein with truncated Pseudomonas Exotoxin: A translocation domain efficiently kills breast cancer cells

“…13,23 The PEA translocation domain has been successfully engineered as a vehicle to deliver therapeutic molecules into cells. [15][16][17][18][19] We have previously constructed a series of genes containing different truncated PEA translocation domains (PEA 253-412aa, PEA 253-364aa and PEA253-358aa) fused with the anti-HER2 single-chain antibody (e23sFv) at the 5'-end, and genes encoding diverse apoptosis-inducing molecules such as caspase-3, 20 caspase-6, 21 truncated AIF 22 and granzyme B 13 at the 3'-end. 14 In this study, we attempted to identify the minimal sequence of the PEA translocation domain for further development in therapeutic drugs.…”

A gene delivery system based on the N-terminal domain of human topoisomerase I

“…5D and G). Unstructured and non-enzymatic functioning TopoN possess DNA binding ability [10], although the details have been unclear. To clarify the binding ability of TopoN, the binding of 6 Â His-PTDTopoNs to different sizes and forms of DNA was assessed.…”

“…Using the virus as a carrier can have higher transfection efficiency, but also with higher potential risk of inflammatory response and virulence. The application limitations of viral vectors are toxicity, restricted targeting of specific cell types [10], limited DNA carrying capacity, production and packaging problems, recombination and expense [11]. The most wildly used of non-viral vectors for delivering DNA into cells are cationic lipids or polymers [12].…”

“…Although the NLSs do not contribute to the catalytic activity, they are essential for the nuclear translocation of the enzyme [36,38]. TopoN also binds to DNA [10,39] without DNA sequence and topological specificity [10].…”