185. Targeted Correction and Restored Function of CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

Abstract

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources – potentially useful for the development of new therapeutic approaches. The primary defect in Cystic Fibrosis (CF), an autosomal recessive disorder, is the regulation of epithelial chloride transport by a chloride channel protein encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recurrent pulmonary infections are responsible for 80 to 90% of the deaths in CF patients. Starting with skin fibroblasts from patients diagnosed with CF, we have derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele vs. the other. Differentiation for a total of 19 days in a protocol designed to derive anterior foregut endoderm, subsequently up-regulated expression of NKX2-1, SOX9, TP63, FOXP2, FOXA2, and CFTR, suggesting commitment of at least a sub-population of cells within the endodermal culture to a lung epithelial cell fate.Once differentiated, mutant CF iPSCs yielded neither mature CFTR protein nor CFTR-specific chloride channel activity (as assayed in Ussing chamber experiments) – whereas corrected CF iPSCs and the normal control WA09 hESCs yielded mature CFTR protein and CFTR-specific chloride channel activity.In vitro differentiation of the mutant CF iPSCs into lung epithelial cells and tissue, controlled for by the parallel differentiation of the otherwise isogenic corrected CF iPSCs, may provide a valuable tool for drug screening and examining the functional consequence of mutant CFTR expression. Furthermore, corrected CF iPSCs present a potential source of patient-specific cells capable, in vitro, of differentiation into various lung stem/progenitor cells – either for transplantation of autologous lung cells or for seeding de-vitalized lung scaffolds ex vivo to generate autologous lungs.