J Biol Chem, Vol. 273, Issue 15, 8835-8841, April 10, 1998
A Modular DNA Carrier Protein Based on the Structure of
Diphtheria Toxin Mediates Target Cell-specific Gene Delivery
Christoph
Uherek
,
Jesús
Fominaya§, and
Winfried
Wels
From the Institute for Experimental Cancer Research,
Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg,
Federal Republic of Germany and the § Department of
Immunology and Oncology, Centro Nacional de
Biotecnología/Consejo Superior de Investigaciones
Científicas, Universidad Autónoma de Madrid, Campus
Cantoblanco, Madrid E-28049, Spain
Modular fusion proteins that combine distinct
functions required for cell type-specific uptake and intracellular
delivery of DNA present an attractive approach for the development of
self-assembling vectors for targeted gene delivery. Here, we describe a
novel DNA carrier protein termed GD5 that mimics the structure of the bacterial diphtheria toxin (DT) and facilitates target cell-specific gene transfer via receptor-mediated endocytosis. GD5 carries at the N
terminus the DNA-binding domain of the yeast transcription factor Gal4,
which is connected to a C-terminal antibody fragment specific for the
tumor-associated ErbB2 antigen via an internal DT translocation domain
as an endosome escape activity. Bacterially expressed GD5 protein
specifically bound to ErbB2-expressing cells and formed protein-DNA
complexes with a luciferase reporter gene construct. These complexes,
after compensation of excess negative charge with
poly-L-lysine, served as a specific transfection
vector for ErbB2-expressing cells. Inhibitors of endosomal
acidification drastically reduced GD5-mediated transfection, indicating
that the DT translocation domain of GD5, similar to the parental toxin, is strictly dependent on the transit through an acidic environment. Our
results suggest that fusion proteins that employ the natural endosome
escape mechanism of bacterial toxins might aid in the development of
efficient nonviral vectors for applications in gene therapy.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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