122. Seamless Correction by Donor DNA of a Class I CFTR Mutation Facilitated by a Double Nicking CRISPR/Cas9 in CF-iPSCs

Abstract

Development of effective gene modification via Homology Directed Repair (HDR) as well as induced pluripotent stem cells (iPSCs) have been significant advances in development of gene- and cell-based therapies for inherited diseases. Patient-specific iPSCs with a corrected disease causing mutation have the potential to functionally repair damaged tissues and organs. Cystic fibrosis (CF) is the most common inherited disease in the Caucasian population with significant multiorgan damage, caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. We and others have shown seamless gene correction of the F508delCFTR mutation, the most common mutation found in CF patients and classified as Class II CFTR mutation, in CF-iPSCs. Correction of the F508del mutation by small/short DNA fragments (SDFs) was enhanced by sequence-specific transcription activator-like effector endonucleases (TALENs). Given that there are> 2,000 mutations associated with CF, the approach used above makes it possible to develop personalized, mutation specific gene and cell therapeutic strategies for CF patients. The studies here describe the generation of iPSCs from a CF patient homozygous for the W1282X, Class I, CFTR mutation and its correction via HDR. As an alternative to using an SDF/TALEN based system described above, the studies described here use a drug selection-based wtCFTR PiggyBac donor DNA system with clustered randomly interspersed short palindromic repeat (CRISPR)/Cas9 nickases targeting CFTR exon23 (the site of the W1282X mutation). CF W1282X/W1282X patient-derived fibroblasts were retrovirally reprogramed with Yamanaka factors (Oct4/Sox2/Klf4/c-Myc) into CF-iPSCs. To correct the W1282X mutation in the CF-iPSCs, a donor DNA comprising the wtCFTR genomic DNA region including exon23, and a CAG-puroΔTK cassette flanked by PiggyBac repeats ending in the TTAA sequence also found in intron 23 of genomic CFTR. Several pairs of CRISPR/Cas9 nickase were also designed to cleave at specific sites adjacent to the W1282X locus. These nickases were assayed for optimal targeting efficiency. The pair that most effectively introduced nicks in double strand DNA were co-transfected with the donor DNA in CF-iPSC. Candidate corrected clones were selected with puromycin, and then assayed by PCR with one primer inside of PiggyBac cassette and the other outside the homology arms in donor DNA 5’ and 3’ of the PiggyBac Cassette. The donor DNA alone did not catalyze site-directed HDR in clones exhibiting puromycin resistance, however, co-transfection of the donor DNA with the optimized CRISPR/Cas9n pairs gave a targeting efficiency of 4.76% (1/21) and 36.6% (15/41) (colonies with HDR/ puromycin resistant colonies) in independent experiments. The drug-resistance cassette in the clones was excised by overexpressing PiggyBac transposase resulting in seamless correction of the CF-iPSCs. This study was supported by grants from Pennsylvania Cystic Fibrosis, Inc, and Cystic Fibrosis Research, Inc, and PPG DK 088760.