Curative Ex Vivo Hepatocyte-Directed Gene Editing in a Mouse Model of Hereditary Tyrosinemia Type 1

Abstract

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). It has been previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small- and large-animal models of HT1. This study hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1, in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of primary hepatocytes, this study utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA (LV-Cas9) and an adeno-associated virus (AAV) vector to deliver a 1.2 kb homology template (AAV-HT). Cells were isolated from Fah mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah cells ex vivo using AAV-HT was completely dependent on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HT were transplanted into syngeneic Fah mice that had undergone a two-thirds partial hepatectomy or sham hepatectomy. Mice were cycled on/off the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) to stimulate expansion of corrected cells. All transplanted mice became weight stable off NTBC. However, a significant improvement was observed in weight stability off NTBC in animals that received partial hepatectomy. After 6 months, mice were euthanized, and thorough biochemical and histological examinations were performed. Biochemical markers of liver injury were significantly improved over non-transplanted controls. Histological examination of mice revealed normal tissue architecture, while immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.

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 ].…”

Mutational spectrum of Mexican patients with tyrosinemia type 1: In silico modeling and predicted pathogenic effect of a novel missense FAH variant

“…In the present study, the patient in whom HT1 was detected at an early stage was promptly treated with nitisinone and showed a positive 1‐year outcome, which is consistent with the successful experiences reported worldwide (Alvarez & Mitchell ). Nevertheless, the development of hepatocellular carcinoma remains a risk in all HT1 patients, thereby indicating the need for promising novel or complementary therapeutic strategies (Aktuglu‐Zeybek, Kiykim, & Cansever, ; VanLith et al, ).…”

In vitro Studies of Transendothelial Migration for Biological and Drug Discovery

“…In addition, these in vitro models can potentially be used to facilitate screening of quality hepatocytes for clinical transplantation. A vast interest lies in gene editing using CRISPR-Cas technology in autologous hepatocytes for gene therapy targeting inherited metabolic diseases affecting the liver, which has been effective in animal models (160)(161)(162) and is the aim for future therapies. The advantage of the gene editing approach is it eliminates the need for immunosuppression and risk of graft rejection as cells isolated from the patient's resected liver are substrates for transplantation or hepatocytes may be edited in vivo to correct a monogenetic disease.…”