Correction of a urea cycle defect after ex vivo gene editing of human hepatocytes

Mihaela Zabulica,Marcello Maresca,Burcu Bestas,Susanna Engberg,Towe Jakobsson,Carl Jorns,Anna Forslöw,Raghuraman C Srinivasan,Georgios Makris,Olav Rooyackers,Christina Hammarstedt,Barry Rosen,Gabriella Allegri,Beat Thöny,Nicole Rimann,Tomas Jakobsson,Johannes Häberle,Ewa Ellis,Natalie R Van Zuydam,Tanja Scherer,Roberto Gramignoli,Stephen C Strom,Mike Firth,Pınar Akçakaya

doi:10.1016/j.ymthe.2021.01.024

Abstract

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient’s own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective.

Highlights

The urea cycle, consisting of six enzymes and two transporters, is localized mainly in the liver and is essential for arginine biosynthesis and the metabolism of ammonia to urea for elimination from the body
The present study was performed to determine whether (1) liver-humanized mice, created through the high-level repopulation of the liver with human hepatocytes isolated from a patient with severe ornithine transcarbamylase deficiency (OTCD), would recreate the phenotype and symptoms of the human genetic disease, and (2) CRISPR-based ex vivo gene editing procedures could correct the mutation and the disease phenotype without causing any deleterious effects or undesired mutagenesis
The intronic OTCD mutation that leads to truncated protein generation was corrected in patient hepatocytes by ex vivo Cas[9] nuclease-mediated editing, which substantially restored normal mRNA splicing and OTC activity

Summary

Introduction

The urea cycle, consisting of six enzymes and two transporters, is localized mainly in the liver and is essential for arginine biosynthesis and the metabolism of ammonia to urea for elimination from the body. The definitive treatment of severe UCD is orthotopic liver transplantation This and other studies indicate that allogeneic hepatocyte transplantation can provide only partial and temporary corrections,[2,3,4] and recent reports suggest that immune-mediated rejection of the donor cells may limit the survival of donor hepatocytes.[5,6] Direct correction of the mutation in a patient’s own hepatocytes would likely avoid the need for immunosuppressive drugs and immune rejection of cell grafts. Recent developments in genome engineering, CRISPR (clustered regularly interspaced short palindromic repeats) technology, have renewed interest in the treatment of genetic disorders through precise gene editing procedures both ex vivo and in vivo.[7,8,9] Autologous and allogeneic cell therapies employing ex vivo CRISPR genome engineering have recently entered clinical trials for hematopoietic stem cell (HSC) transplants to treat beta globinopathies (ClinicalTrials.gov: NCT03655678 and NCT03745287) and chimeric antigen receptor (CAR) T-based anti-cancer therapies of the immune system (ClinicalTrials.gov: NCT03398967, NCT03398967, and NCT0369011)

Methods

Results

Conclusion