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

• Published in: Molecular therapy Vol. 29; no. 5; pp. 1903 - 1917
• Main Authors: Zabulica, Mihaela, Srinivasan, Raghuraman C., Akcakaya, Pinar, Allegri, Gabriella, Bestas, Burcu, Firth, Mike, Hammarstedt, Christina, Jakobsson, Tomas, Jakobsson, Towe, Ellis, Ewa, Jorns, Carl, Makris, Georgios, Scherer, Tanja, Rimann, Nicole, van Zuydam, Natalie R., Gramignoli, Roberto, Forslöw, Anna, Engberg, Susanna, Maresca, Marcello, Rooyackers, Olav, Thöny, Beat, Häberle, Johannes, Rosen, Barry, Strom, Stephen C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 05-05-2021; American Society of Gene & Cell Therapy
• Subjects: CRISPR; ex vivo; FRGN; genome editing; hepatocyte transplantation; liver-humanized mouse; Medicin och hälsovetenskap; Original; primary hepatocytes; urea cycle disorder; genome editing; CRISPR; FRGN; liver-humanized mouse; primary hepatocytes; urea cycle disorder; hepatocyte transplantation; ex vivo
• ISSN: 1525-0016; 1525-0024; 1525-0024
• DOI: 10.1016/j.ymthe.2021.01.024

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.
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The study describes the preclinical investigation of the ex vivo gene editing of hepatocytes isolated from a patient with severe liver disease. The results reveal that gene editing is safe and effective in actual disease-affected human liver cells both in vitro and in vivo in liver-humanized mice.