Correction of Cystic Fibrosis-specific Induced Pluripotent Stem Cells

Cystic Fibrosis (CF), affecting 1 in 3,500 live births in the US, is a disease caused by aberrant expression of the Cystic Fibrosis Transmemebrane Conductance Regulator (CFTR). While a multi-organ disease, CF-related complications and degradation of the lung is the leading cause of mortality. There are treatments to combat treatments but none that are curative. Recent advances in reprogramming and the ability to genetically modify the resulting induced pluripotent stem cells (iPSCs) have provided an alternative to conventional gene therapies thus far attempted for CF. Here we describe the in vitro generation of iPSCs starting from fibroblast cells obtained from a CF patient. These cells, like embryonic stem cells, show evidence of the ability to differentiate into any cell type. Then, using zinc finger nucleases (ZFNs) to induce a double strand break at the CF loci, we facilitated homologous directed repair of the break by providing a donor molecule that would, upon recombination, correct the disease-conferring mutation. In doing so, we observed an unpredicted allele-specific recombination, which could be applicable for gene correction of other diseases like those caused by dominant negative mutations. We interrogated the original CF fibroblast cells, the CF-iPSCs, and corrected iPSCs by whole genome and exome sequencing to evaluate genomic integrity after reprogramming and correction. Furthermore, in vitro differentiation of corrected iPSCs towards lung and thyroid lineages produces cells that express the mature CFTR protein isoform. This is the first report of site-specific correction of the CFTR gene in CF-specific iPSCs