Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

Abstract

SummaryRecently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). 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.

Prospective isolation of NKX2-1–expressing human lung progenitors derived from pluripotent stem cells

“…Prior reports of targeting GFP to the NKX2-1 locus in human PSCs for the derivation of forebrain lineages resulted in NKX2-1 haploinsufficiency (17). Hence, pursuing a strategy designed to retain intact expression of targeted loci without haploinsufficiency ( Figure 1A and Supplemental Figure 1A; supplemental material available online with this article; https:// doi.org/10.1172/JCI89950DS1), we targeted an exon3-2A-GFP cassette to the second intron of NKX2-1 using either transcription activator-like effector nucleases (TALENs) or CRISPR-Cas9 tools deployed in ESCs (H9) or in our previously published iPSC lines: cystic fibrosis patient-specific C17 iPSCs (18) and normal BU3 iPSCs ( Figure 1 and Supplemental Figure 1) (19). The resulting NKX2-1 Figure 1B).…”

“…Previously published PSC lines iPSC17 (18), BU3 (19), and WA09 (H9 ESC) were maintained in feeder-free conditions on Matrigel (Corning) in mTeSR1 (Stem Cell Technologies) and passaged with Gentle Cell Dissociation Reagent (Stem Cell Technologies). In order to generate NKX2-1 GFP reporter PSC lines, TALENs or CRISPR-based technologies were employed as detailed in the supplement to introduce a DNA double-stranded break into the second intron of NKX2-1 (Supplemental Figure 1A).…”

Cystic Fibrosis: The Dawn of a New Therapeutic Era

“…Zinc finger nuclease homology-directed repair works by correcting mutations in the coding region of the CFTR gene by creating a doublestranded DNA break upstream of the defect and combining it with wild-type CFTR DNA (44). This approach has led to wildtype CFTR expression in vitro as well as restoration of CFTR chloride channel function in human bronchial epithelial cells (45). Another genome-editing system, CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPRassociated protein 9), allows for DNA cleavage in a sequence-specific manner and homologous recombination with a corrected endogenous CFTR locus.…”

Application of CRISPR/Cas9 Genome Editing in Genetic Disorders: A Systematic Review Up to Date

“…In cystic fibrosis, DNA, RNA, or proteins can be targeted for the modifications, but at the level of DNA, replacement of mutated CFTR gene with functional CFTR gene could better therapeutical approach. Correction of CFTR mutation in patient derived iPSCs and intestinal stem cell has been done previously using ZFN [43] and CRISPR [42,44]. In these studies, patient derived fibroblasts were reprogrammed to iPSCs and then co-nucleofected with a CRISPR/Cas9-CFTR gRNA cassette in various combinations demonstrated correction of CFTR gene with16.7% efficiency.…”