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

Rui Wei; Jiayin Yang; Chi-Wa Cheng; Wai-In Ho; Na Li; Yang Hu; Xueyu Hong; Jian Fu; Bo Yang; Yuqing Liu; Lixiang Jiang; Wing-Hon Lai; Ka-Wing Au; Wai-Ling Tsang; Yiu-Lam Tse; Kwong-Man Ng; Miguel A Esteban; Hung-Fat Tse

doi:10.1016/j.jhepr.2021.100389

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

JHEP Rep. 2021 Oct 30;4(1):100389. doi: 10.1016/j.jhepr.2021.100389. eCollection 2022 Jan.

Authors

Rui Wei^{1

2

3}, Jiayin Yang^{1

4}, Chi-Wa Cheng^{1

2}, Wai-In Ho^{1

2}, Na Li^{1

2}, Yang Hu^{1

2}, Xueyu Hong¹, Jian Fu⁴, Bo Yang⁴, Yuqing Liu⁴, Lixiang Jiang⁴, Wing-Hon Lai^{1

2}, Ka-Wing Au^{1

2}, Wai-Ling Tsang¹, Yiu-Lam Tse^{1

2}, Kwong-Man Ng^{1

2

3}, Miguel A Esteban^{2

5

6

7}, Hung-Fat Tse^{1

2

3

8}

Affiliations

¹ The Cardiology Division, Department of Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.
² Hong Kong-Guangdong Stem Cell and Regenerative Medicine Research Centre, The University of Hong Kong and Guangzhou Institutes of Biomedicine and Health, Hong Kong, China.
³ Center for Translational Stem Cell Biology, Hong Kong, China.
⁴ Cell Inspire Therapeutics Co., Ltd and Cell Inspire Biotechnology Co., Ltd, Shenzhen 518102, China.
⁵ Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
⁶ Bioland Laboratory (Guangzhou Regenerative Medicine and Health Guangdong Laboratory), Guangzhou 510005, China.
⁷ Joint School of Life Sciences, Guangzhou Medical University and Guangzhou Institutes of Biomedicine and Health, Guangzhou 511436, China.
⁸ Heart and Vascular Center, The University of Hong Kong-Shenzhen Hospital, Shenzhen 518053, China.

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.