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Mol Ther Nucleic Acids. 2016 Jan 5;5:e273. doi: 10.1038/mtna.2015.43.

TALENs Facilitate Single-step Seamless SDF Correction of F508del CFTR in Airway Epithelial Submucosal Gland Cell-derived CF-iPSCs.

Author information

1
Department of Otolaryngology - Head and Neck Surgery, University of California-San Francisco, San Francisco, California, USA.
2
Department of Molecular Medicine, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
3
California Pacific Medical Center Research Institute, San Francisco, California, USA.
4
Childrens Hospital Oakland Research Institute, Oakland, California, USA.
5
Present address: Graduate Program in Biochemistry, Molecular, Cellular, and Developmental Biology, University of California-Davis, Davis, California, USA.
6
Present address: Molecular Department, Hunter Laboratories, Campbell, California, USA.
7
Liver Center, University of California-San Francisco, San Francisco, California, USA.
8
Present address: Heinrich-Heine-Universität Düsseldorf, Institut für Genetik, Düsseldorf, Germany.
9
Present address: Graduate Program in the Department of Biosystems Science and Engineering, ETH, Zürich, Switzerland.
10
Department of Laboratory Medicine, University of California-San Francisco, San Francisco, California, USA.
11
Blood Systems Research Institute, San Francisco, California, USA.
12
Department of Medicine, University of California-San Francisco, San Francisco, California, USA.
13
Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.
14
Department of Pediatrics, Stanford University, Stanford, California, USA.
15
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Helen Diller Family Comprehensive Cancer Center, Institute for Human Genetics, Cardiovascular Research Institute, University of California-San Francisco, San Francisco, California, USA.
16
Department of Pediatrics, University of Vermont College of Medicine, Burlington, Vermont, USA.

Abstract

Cystic fibrosis (CF) is a recessive inherited disease associated with multiorgan damage that compromises epithelial and inflammatory cell function. Induced pluripotent stem cells (iPSCs) have significantly advanced the potential of developing a personalized cell-based therapy for diseases like CF by generating patient-specific stem cells that can be differentiated into cells that repair tissues damaged by disease pathology. The F508del mutation in airway epithelial cell-derived CF-iPSCs was corrected with small/short DNA fragments (SDFs) and sequence-specific TALENs. An allele-specific PCR, cyclic enrichment strategy gave ~100-fold enrichment of the corrected CF-iPSCs after six enrichment cycles that facilitated isolation of corrected clones. The seamless SDF-based gene modification strategy used to correct the CF-iPSCs resulted in pluripotent cells that, when differentiated into endoderm/airway-like epithelial cells showed wild-type (wt) airway epithelial cell cAMP-dependent Cl ion transport or showed the appropriate cell-type characteristics when differentiated along mesoderm/hematopoietic inflammatory cell lineage pathways.

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