Ex Vivo Hepatocyte Reprograming Promotes Homology‐Directed DNA Repair to Correct Metabolic Disease in Mice After Transplantation

Abstract

Ex vivo CRISPR/Cas9‐mediated gene editing in hepatocytes using homology‐directed repair (HDR) is a potential alternative curative therapy to organ transplantation for metabolic liver disease. However, a major limitation of this approach in quiescent adult primary hepatocytes is that nonhomologous end‐joining is the predominant DNA repair pathway for double‐strand breaks (DSBs). This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor HDR after CRISPR/Cas9‐mediated DNA DSBs. Quantitative PCR (qPCR), RNA sequencing, and flow cytometry demonstrated that within 24 hours, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the down‐regulation of hepatocyte function genes, hepatocytes cultured for up to 72 hours could robustly engraft in vivo . To assess functionality long‐term, primary hepatocytes from a mouse model of hereditary tyrosinemia type 1 bearing a single‐point mutation were transduced ex vivo with two adeno‐associated viral vectors to deliver the Cas9 nuclease, target guide RNAs, and a 1.2‐kb homology template. Adeno‐associated viral Cas9 induced robust cutting at the target locus, and, after delivery of the repair template, precise correction of the point mutation occurred by HDR. Edited hepatocytes were transplanted into recipient fumarylacetoacetate hydrolase knockout mice, resulting in engraftment, robust proliferation, and prevention of liver failure. Weight gain and biochemical assessment revealed normalization of metabolic function. Conclusion: The results of this study demonstrate the potential therapeutic effect of ex vivo hepatocyte‐directed gene editing after reprogramming to cure metabolic disease in a preclinical model of hereditary tyrosinemia type 1.

The future of gene-targeted therapy for hereditary tyrosinemia type 1 as a lead indication among the inborn errors of metabolism

“…This method has demonstrated significant efficacy in mouse and pig models of HT1 without increasing the potential for HCC in pigs [ 29 , 37 ]. Ex vivo AAV-based CRISPR/Cas9-mediated gene editing has successfully been achieved in HT1 mouse [ 30 , 32 ]. Additionally, a pig treated ex vivo was maintained for 3 years after dosing, and showed no adverse effects or tumorigenicity [ 29 ].…”

Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases

“…Fah deficiency is fatal owing to the accumulation of toxic metabolites in the hepatic system resulting in cell death, and eventually liver failure [ 41 ] . Ex vivo templated gene correction was enhanced due to an upregulation of HDR-related genes in cultured hepatocytes compared to in vivo counterparts which was further evidenced by an increase in cellular proliferation marker, Ki-67 [ 42 ] . Cultured Fah −/− hepatocytes were treated with a dual AAV gene editing strategy.…”

Ready for Repair? Gene Editing Enters the Clinic for the Treatment of Human Disease

“…This concept was applied to treat hereditary tyrosinemia in a mouse model to correct a point variant in the fumarylacetoacetate hydrolase gene using HDR. 337 A major challenge for this approach is the limited engraftment capacity of hepatocytes in human liver. In cystic fibrosis, investigators are pursuing gene editing of stem cells derived from the airways with the ultimate goal of developing a gene-edited autologous airway stem cell transplantation.…”