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

Firth AL, Menon T, Parker GS, Qualls SJ, Lewis BM, Ke E, Dargitz CT, Wright R1, Khanna A, Gage FH, Verma IM
Source: Cell Rep
Publication Date: (2015)
Issue: 12(9): 1385-1390
Research Area:
Stem Cells
Gene Expression
Basic Research
Cells used in publication:
Induced Pluripotent Stem Cell (iPS), human
Species: human
Tissue Origin:
Nucleofection of iPSC: Adherent cells were dissociated using Tryple (Gibco), centrifuged and resuspended in TeSR containing 10µM ROCK inhibitor, Y27632. 1×106 cells were resuspended in 100µl of Lonza® Nucleofection solution (according to manufacturer’s protocol). The following amounts of DNA were added; Cas9 2µg, gRNA 2 µg, PBHR 5µg, pmaxGFP 2µg, and/or PiggyBac Transposase 2.5µg in the required experimental combination. The samples were then nucleofected using the B-16 protocol on the device. After nucleofection, TeSR was added and cells transferred to a 6-well Matrigel-coated plate. After 24–48 hours of incubation, the nucleofected iPSC were split onto 10cm MEF plates at single-cell density for colony screening or harvested for genomic DNA analysis. PiggyBac Transposase Nucleofection (excision): Excision of the integrated selection cassette from corrected clones was done by overexpressing piggyBac transposase in clonal iPSC lines expressing puromycin resistance and sensitivity to ganciclovir. 4µg of various piggyBac transposase vectors (System Biosciences) were nucleofected into 1×106 iPSC using the Amaxa Nucleofector Device as described above, followed by selection with 3µg/ml ganciclovir to eliminate unexcised cells.
Lung disease is a major cause of death in the United States, with current therapeutic approaches serving only 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 (iPSCs) 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 iPSCs 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.