GENE TARGETING OF CFTR DNA IN CF EPITHELIAL-CELLS

A goal of cystic fibrosis (CF) gene therapy is correction of the mutan t CF transmembrane conductance regulator (CFTR) gene with wild-type (w t) DNA sequences td restore normal CFTR protein and function. Experime nts with wtCFTR cDNA expression vectors have shown that the Cl ion tra nsport phenotype associated with CF can be corrected to resemble that in normal cells. An alternative to cDNA-based gene therapy strategies is one that corrects endogenous mutant sequences by targeted replaceme nt with the wt homologue. To test whether such a strategy was feasible , a small fragment homologous replacement (SFHR) strategy was used to replace specific genomic sequences in human epithelial cells. Small fr agments of genomic wtCFTR DNA were transfected into transformed, CF ep ithelial cells. Replacement by exogenous CFTR DNA at the appropriate g enomic locus and its expression as mRNA was indicated by: (1) allele-s pecific polymerase chain reaction (PCR) amplification of genomic DNA a nd mRNA-derived cDNA; and (2) hybridization of PCR products with allel e-specific probes. In addition, the functional activity of CFTR protei n was determined by whole cell patch clamp. Southern hybridization and patch clamp analysis suggested that approximately 1 in 100 CF cells u nderwent a homologous replacement event that resulted in intact Cl tra nsport.