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

• Published in: Hepatology communications 2019-04, Vol.3 (4), p.558-573
• Main Authors: VanLith, Caitlin J., Guthman, Rebekah M., Nicolas, Clara T., Allen, Kari L., Liu, Yuanhang, Chilton, Jennifer A., Tritz, Zachariah P., Nyberg, Scott L., Kaiser, Robert A., Lillegard, Joseph B., Hickey, Raymond D.
• Format: Article
• Language: English
• Subjects: Cell cycle; CRISPR; Deoxyribonucleic acid; DNA; DNA repair; Drinking water; Epidermal growth factor; Fibroblasts; Flow cytometry; Gene therapy; Genome editing; Genomics; Liver diseases; Metabolic disorders; Mutation; Original; Transplants & implants; Vectors (Biology)
• ISSN: 2471-254X; 2471-254X
• DOI: 10.1002/hep4.1315

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. This study explored the hypothesis that ex vivo hepatocyte culture could reprogram hepatocytes to favor homology directed repair after CRISPR/Cas9‐mediated DNA double strand breaks. Quantitative PCR, RNA sequencing, and flow cytometry demonstrated that within 24h, primary mouse hepatocytes in ex vivo monolayer culture decreased metabolic functions and increased expression of genes related to mitosis progression and HDR. Despite the downregulation of hepatocyte function genes, hepatocytes cultured for up to 72h could robustly engraft in vivo and rescue a mouse model of hereditary tyrosinemia type 1 (HT1) using two adeno‐associated v