Expression of Delta F508 CFTR in normal mouse lung after site-specific modification of CFTR sequences by SFHR

The development of gene targeting strategies for specific modification of g enomic DNA in human somatic cells has provided a potential gene therapy for the treatment of inherited diseases. One approach, small fragment homologo us replacement (SFHR), directly targets and modifies specific genomic seque nces with small fragments of exogenous DNA (400-800 bp) that are homologous to genomic sequences except for the desired modification. This approach ha s been effective for the in vitro modification of exon la in the cystic fib rosis transmembrane conductance regulator (CFTR) gene in human airway epith elial cells. As another step in the development of SFHR for gene therapy st udies were carried out to target and modify specific genomic sequences in e xon 10 of the mouse CFTR (mCFTR) in vivo. Small DNA fragments (783 bp), hom ologous to mCFTR except for a 3-bp deletion (Delta F508) and a silent mutat ion which introduces a unique restriction site (Kpnl), were instilled into the lungs of normal mice using four different DNA vehicles (AVE, LipofectAM INE, DDAB, SuperFect). Successful modification was determined by PCR amplif ication of DNA or mRNA-derived cDNA followed by Kpnl digestion. The results of these studies showed that SFHR can be used as a gene therapy to introdu ce specific modifications into the cells of clinically affected organs and that the cells will express the new sequence.