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.

Keywords: AFP, alpha-fetoprotein; ALB, albumin; ATP7B, ATPase copper transporting beta; ATPase copper transporting beta polypeptide (ATP7B); Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9; EB, embryoid body; RFLP, restriction fragment length polymorphism; Single-stranded Oligodeoxynucleotide (ssODN); TGN, trans-Golgi network; WD, Wilson’s disease; Wilson’s disease; cell therapy; gene correction; iHep(s), iPSC-derived hepatocyte(s); iPSC, induced pluripotent stem cell; iPSC-derived hepatocytes (iHeps); induced pluripotent stem cell (iPSC); sgRNA, single guide RNA; ssODN, single-stranded oligodeoxynucleotide.

Graphical abstract

Fig. 1

Correction of ATP7B R778L in ATP7B^R778L/R778L iPSCs and generation of iHeps from iPSCs. (A) Design of sgRNAs and ssODNs for correction of ATP7B R778L variant (G>T). Arrow heads indicate the cutting sites of sgRNA-1 (blue) or sgRNA-2 (green). Pink shaded areas indicate the R778L variant (in genome) or the corrected sequence (in ssODN). “G” highlighted in green in ssODN indicates the C>G silent mutation site. The pink rectangle in ssODNs indicates the exon region of ATP7B. (B) Sanger sequencing confirmed the correction of R778L mutant (T>G) in ATP7B^R778L/R778L iPSCs. (C) Immunofluorescence staining showed that iHeps highly express hepatic specific markers (ALB, AAT, HNF4A and ASGR1). Scale bar represents 50 μm. (D) ELISA result showed the secreted hALB level in the supernatant of indicated iHeps at day 17 to 18 of differentiation (n = 7). Error bars indicate SEM. hALB, human albumin; iHep(s), iPSC-derived hepatocyte(s); iPSC, induced pluripotent stem cell; sgRNA, single guide RNA; ssODN, single-stranded oligodeoxynucleotide;

Fig. 2

ATP7B^WT/R778L gene-corrected iHeps display normal ATP7B subcellular localization and function. (A) Representative confocal images show different co-localization patterns of ATP7B and P230 in the indicated iHeps in normal culture conditions (baseline) or after 200 μM CuCl₂ treatment for 2 hours (copper overload). Scale bar represents 20 μm. (B) Percentage of ATP7B and P230 co-localization in the indicated iHeps with/without copper treatment (n = 5). Error bars indicate SEM; ∗∗∗p <0.001, p value was obtained using two-way ANOVA. (C) Left panel: Schematic view of the copper-responsive element luciferase reporter assay. Right panel: Line chart shows luminescence induction fold-change of the indicated iHeps after 0 μM, 50 μM, 75 μM and 100 μM CuCl₂ treatment for 24 hours (n = 4). Error bars indicate SEM, p values are indicated on the figure and were obtained using two-way ANOVA. iHep(s), iPSC-derived hepatocyte(s); iPSC, induced pluripotent stem cell; MP, minimal promoter. MRE, metal-responsive element; WT, wild-type.

Fig. 3

Transplantation of iHeps into Atp7b^-/-/ Rag2^-/-/ Il2rg^-/- Wilson’s disease mouse liver. (A) Schematic of the engraftment of iHeps into the liver of ARG mice (Atp7b^-/-/ Rag2^-/-/ Il2rg^-/-). (B) Immunofluorescence staining for hALB shows iHeps incorporated into Wilson’s disease mouse livers at 8 weeks post engraftment. Rectangles indicate the zoom-in areas. Scale bar represents 100 μm. (C) Percentage of human albumin positive cells in mouse livers transplanted with the indicated iHeps, indicating similar engraftment efficiencies between the ATP7B^R778L/R778L and ATP7B^WT/R778L iHeps group. Error bars indicate SEM (n = 6 in Matrigel group, n = 5 in ATP7B^R778L/R778L iHep group, n = 4 in ATP7B^WT/R778L iHep group, and n represents animal number; 2 liver sections and 10 random fields (200x magnification) from each section were calculated for each animal). P values were obtained using one-way ANOVA adjusted with Dunnett’s multiple comparison; error bars indicate SEM. hALB, human albumin; iHep(s), iPSC-derived hepatocyte(s); iPSC, induced pluripotent stem cell; WT, wild-type.

Fig. 4

Transplantation of ATP7B^WT/R778L iHeps attenuates liver injury and reduces hepatic copper content in Wilson’s disease mice. (A) Plasma ALT level in ARG control mice (Atp7b^+/-/ Rag2^-/-/ Il2rg^-/-) or ARG mice (Atp7b^-/-/ Rag2^-/-/ Il2rg^-/-) engrafted with the indicated iHeps at 8 weeks post transplantation. (n = 3 in ARG control mice group or ARG mice transplanted with ATP7B^WT/R778L iHeps group, n = 5 in ARG mice transplanted with Matrigel or ATP7B^R778L/R778L iHeps groups; n represents animal number). (B) Representative liver sections with Picrosirius red staining show the status of liver fibrosis in each group of animals. Scale bar represents 100 μm. (C) Percentage of fibrotic areas calculated according to Picrosirius red staining. (n = 3 in ARG control mice group or ARG mice transplanted with ATP7B^WT/R778L iHeps group, n = 5 in ARG mice transplanted with Matrigel or ATP7B^R778L/R778L iHeps groups, and n represents animal number; 5 different liver sections and 3 random fields (100x magnification) per section were calculated for each animal). (D) Hepatic copper content of the indicated mouse livers, measured by inductively coupled plasma mass spectrometry. (n = 3 in ARG mice transplanted with Matrigel or ATP7B^WT/R778L iHeps groups, n = 5 in ARG control mice or ARG mice transplanted with ATP7B^WT/R778L iHep group, and n represents animal number). (E) Representative images of nuclei staining with DAPI show hepatocyte nucleic structures of liver sections in the indicated mouse group, and cells with nuclear inclusions are indicated with red arrows. Scale bar represents 25 μm. (F) Percentage of cells with nuclear inclusions in each mouse group calculated according to DAPI staining. (n = 3 in ARG control mice group or ARG mice transplanted with ATP7B^WT/R778L iHeps group, n = 5 in ARG mice transplanted with Matrigel or ATP7B^R778L/R778L iHeps groups, and n represents animal number; 2 different sections and 10 random fields (200x magnification) from each section were calculated for each animal). P values were obtained using one-way ANOVA adjusted with Dunnett’s multiple comparison; error bars indicate SEM. ALT, alanine aminotransferase; iHep(s), iPSC-derived hepatocyte(s); iPSC, induced pluripotent stem cell; WT, wild-type.