Efficient drug screening and gene correction for treating liver disease using patient-specific stem cells

Abstract

Patient-specific induced pluripotent stem cells (iPSCs) represent a potential source for developing novel drugand cell- therapies. Although increasing numbers of disease-specific iPSCs have been generated, there has been limited progress in iPSC-based drug screening/discovery for liver diseases, and the low gene targeting efficiency in human iPSCs warrants further improvement. Using iPSC lines from patients with alpha-1 antitrypsin (AAT) deficiency, for which there is currently no drug- or gene- therapy available, we established a platform to discover new drug candidates and to correct disease-causing mutation with a high efficiency. A high-throughput format screening assay based on our hepatic differentiation protocol was implemented to facilitate automated quantification of cellular AAT accumulation using a 96-well immunofluorescence reader. To expedite the eventual application of lead compounds to patients, we conducted drug screening utilizing our established library of clinical compounds, the Johns Hopkins Drug Library, with extensive safety profiles. Through a blind large-scale drug screening, five clinical drugs were identified to reduce AAT accumulation in diverse patient iPSC-derived hepatocyte-like cells. In addition, using the recently developed transcription activator-like effector nuclease (TALEN) technology, we achieved high gene targeting efficiency in AAT-deficiency patient iPSCs with 25–33% of the clones demonstrating simultaneous targeting at both diseased alleles. The hepatocyte-like cells derived from the gene-corrected iPSCs were functional without the mutant AAT accumulation. This highly efficient and cost-effective targeting technology will broadly benefit both basic and translational applications. Conclusions: Our results demonstrated the feasibility of effective large-scale drug screening using an iPSC-based disease model and highly robust gene targeting in human iPSCs; both of which are critical for translating the iPSC technology into novel therapies for untreatable diseases.

Impact of regulatory variation across human iPSCs and differentiated cells

“…While iPSCs have been used to model a number of human diseases (Yagi et al 2011;Choi et al 2013;Liang et al 2013;Miller et al 2013;Aflaki et al 2014;Pashos et al 2017;Cayo et al 2017), there is a limited amount of work demonstrating their ability to model regulatory phenotypes Alasoo et al 2017). iPSC-CMs recapitulate gene expression patterns observed in primary heart tissue obtained from the GTEx Consortium, and eQTLs identified in iPSC-CMs are also enriched among eQTLs identified in primary heart tissue (Supplemental Fig.…”

Mitochondrial Respiratory Defect Causes Dysfunctional Lactate Turnover via AMP-activated Protein Kinase Activation in Human-induced Pluripotent Stem Cell-derived Hepatocytes

“…Hepatic Differentiation-Human iPSCs were differentiated into hepatocytes using protocols previously reported (30,31), with some modifications. The overall procedures are described in Fig.…”

Utilization of TALEN and CRISPR/Cas9 technologies for gene targeting and modification

“…TALENs have been used to correct a mutation found in patients suffering from alpha-1 antitrypsin (AAT) deficiency, a genetic liver disorder that predisposes patients to liver cirrhosis and hepatocellular carcinoma, cystic fibrosis, and Gaucher's disease. 72,73 Moreover, CRISPR/Cas9 has been used to study cystic fibrosis by correcting a mutation found in the transmembrane conductor receptor (CFTR) gene in primary adult stem cells. 74 TALENs and the CRISPR/Cas9 system have also been used to target mutations found in iPS cells derived from Duchenne muscular dystrophy (DMD) patients.…”