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Case Study: Wilson's Disease

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Overview Workflow Model Development Response to drug treatment

Overview

DefinGEN have created Wilson’s disease model hepatocytes for the evaluation of therapeutic candidates. Wilson’s disease is an autosomal-recessive disorder of the copper-transporting gene ATP7B, causing copper deposition and toxicity in the liver, brain, eyes and other organs. Using our in-house iPSC line we generated clones containing several pathogenic mutations of the ATP7B gene including p.H1069Q and p.R778L via CRISPR gene-editing, differentiated to hepatocytes using our Opti-Diff process and then characterised using high content imaging and fluorescence assays.

Disease model hepatocytes were shown to have increased toxicity and oxidative stress resulting from copper challenge when compared to wild-type cells which could be rescued in a dose-dependent manor using the copper-chelator Trientine or a number of function-restoring mRNA therapies.

These data confirm both sensitivity and specificity of response to environmental challenge and rescue treatments, thereby demonstrating that this disease model is highly applicable to candidate drug screening in Wilson’s disease.

Find out more about our disease modeling capabilities >

Workflow

Figure 1. Schematic overview of DefiniGEN's disease modelling platform: introduction of two of the most common genetic mutations within ATP7B associated with Wilson’s disease, iPSC differentiation into hepatocytes, phenotypic validation after copper exposure and chelator treatment.

Model Development - Characterization

Disease circuit verification

Figure 2. Sanger sequencing analysis showing the presence of a homozygous c.2333G>T (CGG>CTG; R778L) mutation in exon 8 of ATP7B gene in WD R778L iPSC model (top) and a homozygous c.3207C>A (CAC>CAA; H1069Q) mutation in exon 14 of ATP7B gene in the WD H1069Q iPSC model (bottom). The rectangles indicate the missense pathogenic mutations.

Characterization Def-HEP ATP7B R778L and ATP7B H1069Q hepatocyte-like cells.

Figure 3. Representative images of ATP7B R778L and ATP7B H1069Q HEPs showing a typical cobblestone morphology. Magnification 100x.

Gene expression analysis

Figure 4. Gene expression analysis of key hepatic markers: ALB, A1AT and HNF4a showing efficient hepatic differentiation ability of WD-introduced iPSCs. PHH cDNA used as the positive control of full hepatic maturation.

Vulnerability of WD-modelled hepatocytes against excessive copper supplementation.

Figure 5. Representative images of WT-, ATP7B R778L- and ATP7B H1069Q hepatocytes treated with different doses of CuCl2 for 24hrs showing disturbed hepatocytes morphology of WD modelled HEPs due to copper induced toxicity. Magnification, 100x.

Cell viability post CuCl2 supplementation measured by CellTiter Glo

Figure 6. Cell viability of WT-, ATP7B R778L- and ATP7B H1069Q hepatocytes 24hr post CuCl2 supplementation measured by CellTiter Glo. Data presented as relatively values to corresponding 0 µM CuCl2 samples, set as 100%.

Defective excretion of intracellular copper ion and high oxidative stress in WD modelled hepatocytes upon copper challenge

Figure 7. Simultaneous live imaging of copper levels and oxidative stress in WT-, ATP7B R778L- and ATP7B H1069Q HEPs treated for 24hrs with escalating doses of CuCl2 (0-500 µM). WD modelled hepatocytes show excessive dose-dependent accumulation of intracellular copper and greater oxidative stress upon copper supplementation compared to control WT hepatocytes.

Coppergreen bio tracker

Figure 8. Quantitative evaluation of copper levels measured using fluorescence induced by Coppergreen dye. Fluorescence intensities normalized to number of nuclei (6 fields).

Response to Trientine Hydrochloride in WD ATP7B H1069Q HEPs exposed simultaneously to Cu ions for 24hrs

Representative images of CellROX Orange staining Large

Figure 9. Representative images of CellROX Orange staining showing a dose-dependent reduction of oxidative stress levels upon Trientine treatment. Images were acquired using CellInsight CX7 HCS instrument and analysed using Thermo Scientific HCS Studio Cell Analysis software.

Dose response

Figure 10. Left) Dose response curve of the effect of Trientine on ROS production, measured with CellROX Orange dye, shows dose-dependent relief of oxidative stress in a range of 100-500 µM. X axis= drug concentration, Y axis= relative fluorescence intensity values normalized to untreated control group, set as 0%. Right) Dose response curve of the effect of Trientine treatment on hepatocytes viability, measured using CellTiter Glo, shows a dose-dependent increase in hepatocytes fitness upon chelator treatment. X axis=drug concentration, Y axis= relative luminescence values normalized to untreated control group, set as 100%.