A FLUORESCENCE VIDEOENDOSCOPY TECHNIQUE FOR DETECTION OF GENE-TRANSFER AND EXPRESSION

The green fluorescent protein (GFP) has previously been adapted as a r eporter for gene transfer and expression in mammalian cells in culture and in tissue sections. Herein is described a new method for detectin g GFP in situ within epithelia accessible to fiberoptic endoscopy by i ncorporating fluorescent filters for detection of GFP into an existing fiberoptic endoscopy system. This device was used to detect expressio n of GFP from adeno-associated virus (AAV; dose of 3 x 10(7) IU) and a denovirus (Ad; dose of 1 of 10(9) to 1 x 10(10) p.f.u.) vectors within the bronchial epithelium of New Zealand white rabbits. GFP expression was confirmed by fluorescence-activated cell sorting (FACS), direct f luorescence microscopy of cytospin preparations of brushed cells, and by fluorescence microscopy of fixed tissue sections. This reporter gen e/detection system was then used to track the time course of expressio n of the AAV vector in the bronchial epithelium over the first 30 days after administration. The transduction frequency in the treated regio n of the epithelium peaked at around 50% at 21 days after transduction . Vector expression was still present at around 20% efficiency at 30 d ays after administration. This example indicates how this method could be used to reliably track gene transfer in living animals or patients .