¹Department of Pediatrics, University of Florida, Gainesville, FL, USA

²Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, FL, USA

³Gene Therapy Center of the University of Florida, Gainesville, FL, USA

⁴Eudowood Division of Pediatric Respiratory Sciences of the Johns Hopkins University, Baltimore, MD, USA

The green fluorescent protein (GFP) has previously been adapted as a reporter for gene transfer and expression in mammalian cells in culture and in tissue sections. Herein is described a new method for detecting GFP in situ within epithelia accessible to fiberoptic endoscopy by incorporating fluorescent filters for detection of GFP into an existing fiberoptic endoscopy system. This device was used to detect expression of GFP from adeno-associated virus (AAV; dose of 3 ´ 10⁷ IU) and adenovirus (Ad; dose of 1 ´ 10⁹ to 1 ´ 10¹⁰ p.f.u.) vectors within the bronchial epithelium of New Zealand white rabbits. GFP expression was confirmed by fluorescence-activated cell sorting (FACS), direct fluorescence microscopy of cytospin preparations of brushed cells, and by fluorescence microscopy of fixed tissue sections. This reporter gene/detection system was then used to track the time course of expression of the AAV vector in the bronchial epithelium over the first 30 days after administration. The transduction frequency in the treated region of the epithelium peaked at around 50% at 21 days after transduction. Vector expression was still present at around 20% efficiency at 30 days after administration. This example indicates how this method could be used to reliably track gene transfer in living animals or patients.

adeno-associated virus (AAV); gene therapy; adenovirus (Ad); green fluorescent protein (GFP); endoscopy; bronchoscopy