Quantitative detection of CFTR mRNA in gene transfer studies in human, murine and simian respiratory tissues in vitro and in vivo

Pre-clinical and clinical studies aimed to correct the basic defect of cystic fibrosis (CF) by transferring the wild type gene into the airway cells have already shown promising results. One of the main unsolved questions of these studies is the quantitation of the amount of gene required to correct the defect. In this respect, suitable technologies able to measure how much gene is actually transferred into the airway cells are under development. In this article we present a series of application of a single-tube competitive RT-PCR assay to quantitate the vector-encoded CFTR mRNA after gene transfer in human, mouse or monkey respiratory cells. These assays are able to measure very low levels of mRNA, with advantages over traditional expression systems for vector-encoded transcript (Northern analysis) or protein (immunocytochemistry). In some instances they may indicate directly the ratio of vector-encoded versus endogenous transcript, being applicable with both viral-derived and synthetic vectors. The assays presented here are instrumental for future application in gene delivery pre-clinical and clinical trials designed to identify corrective doses of the vector, by providing the basic information on the amount of vectorencoded CF gene transcript expressed in the airway cells.