Autologous Gene and Cell Therapy Provides Safe and Long-Term Curative Therapy in A Large Pig Model of Hereditary Tyrosinemia Type 1

Abstract

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson’s trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

Hepatotoxicity and Toxicology of In Vivo Lentiviral Vector Administration in Healthy and Liver-Injury Mouse Models

“…25 In one longterm pig from our ex vivo study, no tumorigenicity was present in the liver or elsewhere after 3 years post-transplant. 26 Finally, the use of a liver-specific promoter further focusses any potential for genotoxicity from transactivation to hepatocytes, where screening for tumorigenesis is already part of the clinical management of human disease via frequent imaging.…”

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

“…HT1 has been experimentally targeted by ex vivo gene delivery approaches where hepatocytes are collected from a syngeneic donor (mouse) [ 41 ] or via laparoscopic partial resection of the liver in the actual test animal (pig) [ 29 , 42 ]. These hepatocytes are cultured and transduced with LV vectors carrying a wildtype FAH to rescue normal function and cure the disease [ 40 ].…”

“…These hepatocytes are cultured and transduced with LV vectors carrying a wildtype FAH to rescue normal function and cure the disease [ 40 ]. 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 ].…”

“…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 ]. An alternative approach designed to disrupt the HPD gene (the target of NTBC therapy) by using similar gene editing methods showed efficacy in treating test animals, but this results in subjects with a second homozygous or compound heterozygous genetic mutation, which results in sustained HT1 and HT3 phenotypes [ 43 ].…”

The concept of “domino” in liver and hepatocyte transplantation

“…These hepatocytes were re-transplanted to the same pigs after using ex vivo gene therapy with lentiviral vectors, and the short- and long-term results showed the stable curative efficacy of genetically modified hepatocytes. 61 Nevertheless, the effectiveness and safety of genetically corrected hepatocytes as well as the long-term metabolic consequences still require further confirmation before their application in clinical practice. In contrast, allogeneic hepatocyte transplantation may be more reliable in clinical practice.…”