Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease

Abstract

Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH+/− pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH−/− pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopthy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH−/− pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH−/− pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.

Chronic Phenotype Characterization of a Large-Animal Model of Hereditary Tyrosinemia Type 1

“…13,14 Genotyping of all pigs was performed by established PCR and Southern blot assays using ear and tail tissue.…”

“…Animals in group 3 had overlap with our former study. 13,14 Weight Follow-Up All animals were weighed daily for the first month, twice weekly up to 3 months of age, and then weekly to the end of the study. Weight gain in kg per month was determined for each pig, and the average was calculated for all pigs in one group.…”

“…FAH immunohistochemistry was performed by using a polyclonal rabbit anti-FAH primary antibody, as previously described. 14,20 Fibrosis was graded with the modified METAVIR classification scheme, as previously described. 21 …”

“…12 To address the lack of a preclinical model of FAH deficiency, a novel large-animal model was generated by adeno-associated virus-mediated gene knockout and outbreeding of the produced FAH þ/À animals to generate homozygote FAH À/À pigs. 13,14 Because of their similarity in size, anatomy, and overall metabolic activity to humans, pigs offer improved models for translational biomedical research. 15e17 As described previously, FAH À/À pigs provide a clinically relevant model of the acute HT1 phenotype.…”

“…15e17 As described previously, FAH À/À pigs provide a clinically relevant model of the acute HT1 phenotype. 14 This study characterizes the chronic phenotype of HT1 in a large-animal model. Given a low maintenance dose of NTBC, FAH À/À pigs demonstrated growth retardation and biochemical abnormalities, such as elevated liver enzymes, tyrosine, succinylacetone (SUAC), and a-fetoprotein (AFP).…”