CRISPR-targeted genome editing of human induced pluripotent stem cell-derived hepatocytes for the treatment of Wilson’s disease

Abstract

Background & Aims Wilson’s disease (WD) is an autosomal recessive disorder of copper metabolism caused by loss-of-function mutations in ATP7B , which encodes a copper-transporting protein. It is characterized by excessive copper deposition in tissues, predominantly in the liver and brain. We sought to investigate whether gene-corrected patient-specific induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps) could serve as an autologous cell source for cellular transplantation therapy in WD. Methods We first compared the in vitro phenotype and cellular function of ATP7B before and after gene correction using CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs) in iHeps (derived from patients with WD) which were homozygous for the ATP7B R778L mutation (ATP7B R778L/R778L ). Next, we evaluated the in vivo therapeutic potential of cellular transplantation of WD gene-corrected iHeps in an immunodeficient WD mouse model ( Atp7b -/- / Rag2 -/- / Il2rg -/- ; ARG). Results We successfully created iPSCs with heterozygous gene correction carrying 1 allele of the wild-type ATP7B gene (ATP7B WT/- ) using CRISPR/Cas9 and ssODNs. Compared with ATP7B R778L/R778L iHeps, gene-corrected ATP7B WT/- iHeps restored i n vitro ATP7B subcellular localization, its subcellular trafficking in response to copper overload and its copper exportation function. Moreover, in vivo cellular transplantation of ATP7B WT/- iHeps into ARG mice via intra-splenic injection significantly attenuated the hepatic manifestations of WD. Liver function improved and liver fibrosis decreased due to reductions in hepatic copper accumulation and consequently copper-induced hepatocyte toxicity. Conclusions Our findings demonstrate that gene-corrected patient-specific iPSC-derived iHeps can rescue the in vitro and in vivo disease phenotypes of WD. These proof-of-principle data suggest that iHeps derived from gene-corrected WD iPSCs have potential use as an autologous ex vivo cell source for in vivo therapy of WD as well as other inherited liver disorders. Lay summary Gene correction restored ATP7B function in hepatocytes derived from induced pluripotent stem cells that originated from a patient with Wilson’s disease. These gene-corrected hepatocytes are potential cell sources for autologous cell therapy in patients with Wilson’s disease.

Case report: Treatment of Wilson’s disease by human amniotic fluid administration

“…Therefore, novel therapeutic approaches were warranted to improve the management of neuropsychiatric symptoms. In this context, several animal studies have generated promising results ( 6 , 11 , 12 ). However, there are no ongoing reported clinical studies in neurological-type WD patients.…”

ATP7B gene therapy of autologous reprogrammed hepatocytes alleviates copper accumulation in a mouse model of Wilson’s disease

“…This unsatisfying result was also supported by previous studies. [ 19 , 20 , 21 , 22 , 23 ] The low repopulation efficiency for HLCs transplantation was possibly due to the immature state of hepatic differentiation. Other candidates of cell sources like hepatic transdifferentiation from somatic cells and immortalization of hepatocytes show safety issues like oncogenic transformation or other unpredictable risks.…”

Engineered Platforms for Maturing Pluripotent Stem Cell–Derived Liver Cells for Disease Modeling

“… 93 Another group showed that gene-corrected HLCs from patients with Wilson disease could slow the manifestation of Wilson disease in a mouse model following implantation. 94 …”