TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy.

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing.