A modular DNA carrier protein based on the structure of diphtheria toxin mediates target cell-specific gene delivery.
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.
Uherek
C
C
Institute for Experimental Cancer Research, Tumor Biology Center, Breisacher Strasse 117, D-79106 Freiburg, Federal Republic of Germany.
Fominaya
J
J
Wels
W
W
D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001943:Breast Neoplasms; D019556:COS Cells; D002738:Chloroquine / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D018942:Macrolides; D008744:Methylamines / Q000494:pharmacology; D009550:Nigericin / Q000494:pharmacology; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D057127:Single-Chain Antibodies; D014162:Transfection / Q000187:drug effects / Q000379:methods*; D014407:Tumor Cells, Cultured
D000643:Ammonium Chloride; D000900:Anti-Bacterial Agents; D002738:Chloroquine; D004167:Diphtheria Toxin; D018942:Macrolides; D008744:Methylamines; D009550:Nigericin; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; C111860:GD5 protein, recombinant; C040929:bafilomycin A1; C027451:methylamine