Gene Transfer Using a Novel Fusion Protein, GAL4/Invasin

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Abstract
The delivery of DNA to target cells using simple, defined, nonviral systems has become an area of intense interest in gene therapy. We describe here the development and characterization of one such novel system. A recombinant, bifunctional, fusion protein was expressed and purified from Escherichia coli. This protein consists of the DNA-binding domain of the yeast transcription factor GAL4 fused to the cell binding, internalization domain of the Yersinia pseudotuberculosis inv gene product, invasin. This protein, GAL4/Inv, together with poly-l-lysine, formed complexes with a chloramphenicol acetyltransferase (CAT) reporter plasmid that contains eight repeats of the GAL4 consensus recognition sequence. These complexes were shown to transfect target cells in an invasin receptor-dependent manner, resulting in transient CAT expression. A simple, targeted DNA delivery vehicle, as we describe here, represents a viable approach to nonviral gene delivery. In an effort to address the various technical and regulatory concerns associated with any gene delivery vehicle, the development of defined and easily characterized systems has taken on great significance. The ability to engineer, express, and purify recombinant proteins with specific functions represents a viable means by which to address these concerns. We describe here the generation of such a system that consists of only three parts: a single fusion protein, a DNA condensation agent, and plasmid DNA. In the specific system characterized here, the protein is a fusion between domains of GAL4 and invasin, the DNA condensation agent is poly-l-lysine, and the plasmid is a chloramphenicol acetyltransferase (CAT) expression vector that harbors eight repeats of the GAL4 consensus recognition sequence. This simple system was able to transfect target cells effectively in a receptor-specific manner, resulting in transient gene expression. The potential utility and appeal of this gene delivery system lie with its modularity, the ability to control the nature of its constituent parts, and ease of generation.