RESUMO
BACKGROUND/PURPOSE: Since the discovery of the cystic fibrosis transmembrane regulator (CFTR) gene, cystic fibrosis has been an attractive target for gene therapy. Postnatal gene transfer in the respiratory epithelium has been difficult and particularly inefficient in the submucosal gland cells, the target cells for CFTR gene transfer. The authors hypothesized that during development, there is a favorable environment for fetal gene therapy with fewer physical barriers to efficient gene transfer and more accessible epithelial and submucosal gland precursor cells. The authors tested this hypothesis in a novel human fetal tracheal organ culture system using a serotype 2 recombinant AAV that contains an enhanced green fluorescent protein (eGFP) reporter gene (AAV-CMV-eGFP). METHODS: Human fetal tracheas at between 16 and 20 weeks' gestation age were used in this study. The distal end of each trachea was ligated and secured in an upright position with the open proximal end facing up. Only the ante-lumenal surface was exposed to culture media. 5 x 10(9) particles of AAV-CMV-eGFP were administered intratracheally through the open end. Fetal tracheas were maintained in tracheal organ culture media and harvested at either 2 weeks (n = 3) or 4 weeks (n = 7) after injection. The fetal tracheas were processed for routine H&E, standard electron microscopy (EM), and fluorescence microscopy for analysis of eGFP transgene expression. RESULTS: Histology confirmed the preservation of structural integrity out to 4 weeks of fetal tracheal organ culture. EM showed intact tight junctions of the apical respiratory epithelium. At 2 weeks after AAV-CMV-eGFP injection, there was minimal transgene expression. However, at 4 weeks there was extensive transgene expression in not only the respiratory surface epithelium but also the submucosal gland cells of the human fetal tracheal organ culture. Transgene expression was seen in nearly all cells in the submucosal glands. CONCLUSIONS: AAV-mediated gene transfer in human fetal tracheal organ culture was highly efficient with excellent transgene expression at 4 weeks in both respiratory surface epithelium and submucosal gland cells. This highly efficient gene transfer may result from fewer physical barriers and more accessible target precursor cells. These results are consistent with more efficient gene transfer in fetal tracheobronchial epithelium and show the potential for fetal gene therapy using AAV for the treatment of congenital airway disease such as cystic fibrosis.