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

Abstract

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.

Expression of Endogenous Angiotensin-Converting Enzyme 2 in Human Induced Pluripotent Stem Cell-Derived Retinal Organoids

“…Induced pluripotent stem cell (iPSC) technologies and patient-derived iPSCs provide opportunities for regenerative medicine, drug discovery, and disease modeling from patient-derived stem cells, such as in cystic fibrosis, Huntington’s, Duchenne muscular dystrophy, Alzheimer, amyotrophic lateral sclerosis, age macular degeneration, and retinitis pigmentosa [ 10 , 11 , 12 , 13 , 14 , 15 , 16 ]. Although monolayer cultures are relatively easy to be cultivated, the features that they do not display, the complex tissue organization, limits their bioavailability.…”

hPSC-derived organoids: models of human development and disease

“…In addition, hPSCs are amenable to genome engineering and provide an opportunity to derive iPSC lines from patients carrying disease-associated mutations, making hPSC-derived organoids particularly well suited for modeling genetic disease. iPSC lines derived from patients have been shown to recapitulate pathologic phenotypes in retinal [108], lung [20,66,78], kidney [21], intestinal [109], and colonic [110,111] organoids, among many others. In many of these studies, genome engineering is applied to generate mutation-corrected iPSC lines, which permit the comparison of organoid development between genetic backgrounds that only differ at the site of the mutation under investigation.…”

Treatment of Cystic Fibrosis: From Gene- to Cell-Based Therapies

“…A similar study used ZFNs to correct Phe508del and demonstrated restored CFTR protein expression and function in air-liquid interface cultures established from the edited basal cells ( Suzuki et al, 2020 ). TALEN technologies have also been used to restore normal CFTR expression and activity in organoids derived from Phe508del patient-derived iPSCs ( Fleischer et al, 2020 ). The ability to achieve CFTR correction in additional stem/progenitor cells such as airway basal cells, either as primary airway basal cells or those derived from iPSCs, has specific relevance for progress toward cell-based therapies for CF.…”