Targeted gene delivery to mammalian cells by filamentous bacteriophage.
We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.
D001435:Bacteriophages / Q000235:genetics*; D001943:Breast Neoplasms / Q000473:pathology; D002460:Cell Line; D003110:Colonic Neoplasms / Q000473:pathology; D004705:Endocytosis; D005260:Female; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D018734:Genes, erbB-2; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008297:Male; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011971:Receptors, Immunologic / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000502:physiology; D014162:Transfection / Q000379:methods*