Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.
Bottom Line: Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines.We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other.The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.
Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.Show MeSH
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Mentions: In our targeted correction of the ΔI507/ΔF508 clone 17 iPSCs, we observed a strong preference for targeting the ΔI507 allele versus the ΔF508 allele; sequencing of the unmodified allele yielded either the ΔI507 mutant allele (present in 3 of 21 clones) or ΔF508 mutant allele (in 18 of 21 clones) (a ΔI507/ΔF508 targeting ratio of 6:1) (Figure 4A). Although we initially speculated that perhaps a greater level of chromatin accessibility or transcriptional activity for one allele versus the other may have been responsible, our sequencing of CFTR cDNA from the original ΔI507/ΔF508 iPSCs, as shown previously, revealed transcriptional activity from both mutant alleles (Figure 2D).
Affiliation: Center for Stem Cell and Regenerative Medicine, Brown Foundation Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030, USA.