title: Targeted Integration of a Super-Exon into the CFTR Locus Leads to Functional Correction of a Cystic Fibrosis Cell Line Model.
abstract: In vitro disease models have enabled insights into the pathophysiology of human disease as well as the functional evaluation of new therapies, such as novel genome engineering strategies. In the context of cystic fibrosis (CF), various cellular disease models have been established in recent years, including organoids based on induced pluripotent stem cell technology that allowed for functional readouts of CFTR activity. Yet, many of these in vitro CF models require complex and expensive culturing protocols that are difficult to implement and may not be amenable for high throughput screens. Here, we show that a simple cellular CF disease model based on the bronchial epithelial ΔF508 cell line CFBE41o- can be used to validate functional CFTR correction. We used an engineered nuclease to target the integration of a super-exon, encompassing the sequences of CFTR exons 11 to 27, into exon 11 and re-activated endogenous CFTR expression by treating CFBE41o- cells with a demethylating agent. We demonstrate that the integration of this super-exon resulted in expression of a corrected mRNA from the endogenous CFTR promoter and used short-circuit current measurements in Ussing chambers to corroborate restored ion transport of the repaired CFTR channels. In conclusion, this study proves that the targeted integration of a large super-exon in CFTR exon 11 leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5' end of exon 11.
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg, Germany.
authors.forename: Christien
authors.initials: C
authors.lastname: Bednarski
authors.affiliation: Institute of Animal Physiology, Westphalian Wilhelms-University, Muenster, Germany.
authors.forename: Katja
authors.initials: K
authors.lastname: Tomczak
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg, Germany.
authors.forename: Beate
authors.initials: B
authors.lastname: Vom Hövel
authors.affiliation: Institute of Animal Physiology, Westphalian Wilhelms-University, Muenster, Germany.
authors.forename: Wolf-Michael
authors.initials: WM
authors.lastname: Weber
authors.affiliation: Institute for Cell and Gene Therapy, Medical Center-University of Freiburg, Freiburg, Germany.|Center for Chronic Immunodeficiency, Medical Center-University of Freiburg, Freiburg, Germany.|Faculty of Medicine, University of Freiburg, Freiburg, Germany.
authors.forename: Toni
authors.identifier: http://orcid.org/0000-0002-7757-4630
authors.initials: T
authors.lastname: Cathomen
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D003550:Cystic Fibrosis / Q000235:genetics / Q000473:pathology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D003851:Deoxyribonucleases / Q000737:chemistry / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005091:Exons / Q000235:genetics*; D000072669:Gene Editing / Q000379:methods*; D056426:Genetic Loci / Q000235:genetics*; D005838:Genotype; D006801:Humans; D009154:Mutation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D003851:Deoxyribonucleases