A chimeric fusion protein containing transforming growth factor-α mediates gene transfer via binding to the EGF receptor

Fusion proteins engineered to incorporate distinct functions which co-operate in mediating the cell-type specific uptake and intracellular delivery of DNA present an attractive approach for the development of self-assembling vector systems for targeted gene transfer. Here we have chosen the EGF receptor overexpressed in many human tumors of epithelial origin as a target for a novel modular fusion protein. We have fused a cDNA fragment of the human EGF receptor ligand TGF-alpha to sequences encoding the translocation domain of Pseudomonas exotoxin A as an endosome escape activity, and the DNA-binding domain of the yeast GAL4 transcription factor Upon bacterial expression, this TEG fusion protein displayed specific binding to EGF receptors. Complexes of the chimeric protein and plasmid DNA carrying a luciferase reporter gene, after condensation with poly-L-lysine resulted in an up to 150-fold increase in reporter gene expression in EGF receptor expressing cells in comparison to poly-L-lysine-DNA complexes alone. While in COS-1 cells no additional endosome escape activity was required, in A431 cells gene delivery was dependent on the simultaneous presence of the endosome destabilizing reagent chloroquine indicating that cell-type specific factors such as different intracellular routing of protein-DNA complexes greatly influence transfection efficiency.