iPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.We thank M. Camarasa for her help in HR experiments and Victor Gálvez for his assistance with Figures 1 and 2. Additionally, we thank Patricia Tortosa for providing invaluable technical support along the project. We acknowledge Hubrecht Organoid Technology (https:// hub4organoids.eu/) for providing the rectal organoids from healthy-donor and p.F508del/p.508del patients. We also thank the Beekman’s group for sharing their expertise in human intestinal orga- noids. Finally, we are indebted to Ludovic Vallier for sharing with us the pMCS-AAT-PB:PGKpuroDtk plasmid. This work was supported by grants from the Spanish Ministry for Science and Innovation (PLE2009-0091, RTC-2014-2207-1, and IPT-2011-1402-900000); IS- CIII PI14/01073, cofunded by ERDF/ESF “Investing in your future”; the Balearic Government (16023/2008); the Spanish Cystic Fibrosis Federation (Pablo Motos Grant); Federación ASEM-Telemaratón “Todos somos raros, todos somos únicos”; Fundación Salud 2000; the European Commission (H2020, PHC-667079); and an endow- ment from METROVACESA. J.M.M.-F. was a postdoctoral Berri- kertu fellow granted by the Basque Government. A.F. was a recipient of Juan de la Cierva (JCI-2006-2675) and Torres Quevedo (PTQ-16- 08496) postdoctoral fellowships from the Spanish Ministry for Sci- ence and Innovation