Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs

Abstract

SUMMARY Lung disease is a major cause of death in the USA, with current therapeutic approaches only serving to manage symptoms. The most common chronic and life-threatening genetic disease of the lung is Cystic fibrosis (CF) caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). We have generated induced pluripotent stem cells (iPSC) from CF patients carrying a homozygous deletion of F508 in the CFTR gene, which results in defective processing of CFTR to the cell membrane. This mutation was precisely corrected using CRISPR to target corrective sequences to the endogenous CFTR genomic locus, in combination with a completely excisable selection system which significantly improved the efficiency of this correction. The corrected iPSC were subsequently differentiated to mature airway epithelial cells where recovery of normal CFTR expression and function was demonstrated. This isogenic iPSC-based model system for CF could be adapted for the development of new therapeutic approaches.

Modeling Development and Disease with Organoids

“…The corrected iPS cells were subsequently converted to mature airway epithelial cells demonstrating recovery of normal CFTR function (Firth et al, 2015).…”

Impact of gene editing on the study of cystic fibrosis

“…Direct repair of the ∆F508 mutation in iPS cells using ZFNs (Crane et al 2014), followed by differentiation of corrected cells into epithelia, resulted in the full restoration of Cl − channel function, though the excision of the selection marker to enrich edited cells left a single 34-bp loxP site in the genome. Truly scarless gene editing of CFTR in iPS cells was achieved using Cas9/gRNA and a selection marker flanked by PiggyBac transposon sequences (Firth et al 2015), whereas a cyclic enrichment strategy involving allele-specific PCR enabled the isolation of edited cells generated using SDFs with TALENs with significantly fewer genetic manipulations . Upon differentiation, these cells also showed fully restored CFTR function as assessed by several different assays.…”

Fixing Stem Cells Via Genome Editing: Hope for Cystic Fibrosis?

“…The design and assessment of CFTR-specific nucleases also has been reported previously, including repair of the mutant CFTR gene [6][7][8][9][10]. There are now several classes of sequence-specific nucleases available (ZFNs, TALENs, CRISPR/Cas9, Meganucleases) for DNA sequence-specific gene editing.…”