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

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.

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins Results in High Levels of Gene Editing in Primary Hepatocytes

“…demonstrated the transplantation of patient-derived donor human hepatocytes into FRGN mice to generate a humanized model of ornithine transcarbamylase deficiency. 39 The weakness of this approach is that the chimeric humanized-liver FRGN mice are immune compromised, which limits the type of experiments that can be conducted.…”

“…Primary human hepatocytes were electroporated using modified procedures described in Zabulica et al . 39 Briefly, hepatocytes were electroporated with P3 Primary Cell 4D-Nucleofector solution (Lonza), program CA-137, and the following electroporation conditions: 100 μL P3 nucleofection buffer, 1.8 μL of 20 μg/μL sgRNA, 5 μL of 61 μM V3 SpCas9 (product code: 1081059; Integrated DNA Technologies) or 5 μL of 61 μM V3 HiFi SpCas9 (product code: 1081061; Integrated DNA Technologies), and 3 μL of 100 μM Electroporation Enhancer (product code: 1075916; Integrated DNA Technologies).…”

G Protein-Coupled Receptor GPR35 Suppresses Lipid Accumulation in Hepatocytes

“…Generation of Genome-Edited HepG2 GPR35 KO Cells. Expression of full-length mRNA from the GPR35 gene was eliminated using a dual synthetic gRNA/RNP approach 43 with one guide cutting the intron and one in the exon region to generate a nonfunctional GPR35 protein. The gRNAs were designed with the CRISPR Finder (Welcome Sanger Institute).…”

Primary human hepatocyte gene editing: Prometheus’ chains are loosening

“…Two major roadblocks that have limited clinical progress with hepatocyte transplantation are the lack of renewable high-quality human hepatocyte sources and the inability to efficiently correct inborn errors in PHHs. In this issue of Molecular Therapy, Zabulica et al 3 show the first genetic correction of an inborn error in PHHs.…”

“…Ornithine transcarbamylase (OTC) deficiency is a rare X-linked disorder of the urea cycle, resulting in ammonia elevation with life-threatening neurological complications. Zabulica et al 3 identified a splice acceptor site variant that results in an early stop codon and dysfunctional protein in an OTC patient who underwent liver transplantation. Using synthetic guide RNA and recombinant Cas9 ribonucleoprotein complex transfection, the authors were able to remove the splice variant in 60%-80% of PHHs isolated from the explanted liver and restore OTC function.…”