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

Shingo Suzuki; R Geoffrey Sargent; Beate Illek; Horst Fischer; Alaleh Esmaeili-Shandiz; Michael J Yezzi; Albert Lee; Yanu Yang; Soya Kim; Peter Renz; Zhongxia Qi; Jingwei Yu; Marcus O Muench; Ashley I Beyer; Alessander O Guimarães; Lin Ye; Judy Chang; Eli J Fine; Thomas J Cradick; Gang Bao; Meghdad Rahdar; Matthew H Porteus; Tsuyoshi Shuto; Hirofumi Kai; Yuet W Kan; Dieter C Gruenert

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

Mol Ther Nucleic Acids. 2016 Jan 5;5(1):e273. doi: 10.1038/mtna.2015.43.

Authors

Shingo Suzuki^{1

2}, R Geoffrey Sargent^{1

3}, Beate Illek⁴, Horst Fischer⁴, Alaleh Esmaeili-Shandiz¹, Michael J Yezzi^{1

3}, Albert Lee^{1

5}, Yanu Yang^{3

6}, Soya Kim^{7

8}, Peter Renz^{1

3

9}, Zhongxia Qi¹⁰, Jingwei Yu¹⁰, Marcus O Muench^{10

7

11}, Ashley I Beyer¹¹, Alessander O Guimarães¹¹, Lin Ye¹², Judy Chang¹², Eli J Fine¹³, Thomas J Cradick¹³, Gang Bao¹³, Meghdad Rahdar¹⁴, Matthew H Porteus¹⁴, Tsuyoshi Shuto², Hirofumi Kai², Yuet W Kan^{12

15}, Dieter C Gruenert^{1

3

15

16}

Affiliations

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.

Abstract

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