br Conclusions The following are the supplementary data rela

Conclusions The following are the supplementary data related to this article.
Acknowledgments
Introduction Hereditary tyrosinemia type I (HT1; OMIM #276700) is an autosomal-recessive inborn error of metabolism caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism (de Laet et al., 2013; Grompe, 2001; Sniderman King et al., 1993; Lindblad et al., 1977). The absence of FAH causes accumulation of the toxic metabolite fumarylacetoacetate (FAA) in hepatocytes and renal proximal tubules, the two major cell types that express FAH (Endo and Sun, 2002; Jorquera and Tanguay, 1997; Kubo et al., 1998). Clinically, individuals with HT1 commonly develop symptoms within the first few weeks of life; however, presentation is often variable, even within a family (Sniderman King et al., 1993). Acute onset of HT1 is characterized by severe liver involvement (Russo and O\'Regan, 1990), most frequently leading to death, if untreated. The most common treatment for HT1 is a low-tyrosine diet combined with administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) (Lindstedt et al., 1992), a potent inhibitor of 4-hydroxyphenylpyruvate dioxygenase (Fig. S1). Two Fah-knockout mouse models have been previously described: the c14CoS albino mouse and the FahΔexon5 mouse (Gluecksohn-Waelsch, 1979; Russell et al., 1979; Grompe et al., 1993). Fah-knockout mice have proven a tremendous resource for translational research related to treatment of a metabolic liver disease by various cell and gene therapy approaches (Overturf et al., 1996; Paulk et al., 2010; Lisowski et al., 2012; Huang et al., 2011; Zhu et al., 2014). However, as has been demonstrated elegantly by the creation of ep4 antagonist transmembrane conductance regulator (CFTR) knockout pigs (Rogers et al., 2008), the pig is a more appropriate research model because of its similarity in size, anatomy, and biology to the human (Cooper et al., 2002). We have previously reported the generation and characterization of heterozygous FAH+/− pigs (Hickey et al., 2011) by using adeno-associated virus (AAV) and homologous recombination to target and disrupt the porcine FAH gene, located on chromosome 7 in the pig genome. An AAV vector was used to deliver a knockout construct targeted to exon 5 of FAH fetal pig fibroblasts with an average knockout targeting frequency of 5.4% achieved. Targeted FAH+/− fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes, and multiple viable FAH+/− pigs were born. FAH+/− pigs were phenotypically normal, but had decreased FAH transcriptional and enzymatic activity compared to FAH+/+ animals. Therefore, the goal of this study was to generate and characterize FAH−/− pigs in order to develop a more relevant preclinical model of an inborn error of metabolism than currently exists.
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Discussion As limitations in murine models have become more apparent, a substantial need exists for the creation of improved models of human disease (Prather et al., 2013). The advent of SCNT and improved gene targeting strategies have made the pig the preferred choice for generating large animal models of human diseases (Rogers et al., 2008; Renner et al., 2010; Saeki et al., 2004). Metabolic diseases, or inborn errors of metabolism, are caused by single gene defects in which the absence or dysfunction of a protein results in abnormal synthesis or metabolism of a protein, carbohydrate, or fat. While the individual incidence of each disorder is rare, it is estimated that 10% of all pediatric liver transplants are resultant from inborn errors of metabolism (Hansen and Horslen, 2008). A potential alternative strategy to liver transplantation is hepatocyte transplantation (Fisher and Strom, 2006). Since initial preclinical experiments in a rodent model for Crigler–Najjar syndrome type 1 (Matas et al., 1976), a number of small animal models of metabolic liver diseases have been treated by hepatocyte transplantation, including hereditary tyrosinemia type 1 (Overturf et al., 1996), phenylketonuria (Hamman et al., 2005), Wilson\'s disease (Yoshida et al., 1996), progressive familial intrahepatic cholestasis (De Vree et al., 2000), and alpha-1 antitrypsin deficiency (Overturf et al., 1996; Matas et al., 1976; Wilson et al., 1988). These experiments in rodents have paved the way for hepatocyte transplantation in humans, including the first published partial correction in a patient with metabolic liver disease in 1998 (Fox et al., 1998). A major limitation of hepatocyte transplantation in humans has been a shortage of transplantable cells. Therefore, there has been intense interest in developing new methods to generate hepatocytes from various cell types including fetal and adult liver stem cells (Huch et al., 2013; Schmelzer et al., 2007; Haridass et al., 2009), pluripotent stem cells (Yamada et al., 2002; Yu et al., 2012; Si-Tayeb et al., 2010), and by direct reprogramming from other lineages (Huang et al., 2011; Zhu et al., 2014). The Fah-knockout mouse has played a critical role in many of these studies by providing an essential test of in vivo efficacy of hepatocyte function and repopulation competency (Azuma et al., 2007). However, as has been demonstrated recently by generation of CFTR knockout animals (Rogers et al., 2008), pigs provide a more clinically-relevant model of human disease and should accelerate development of regenerative therapies for various human disorders. We believe, therefore, that a large animal knockout model of FAH-deficiency will provide an important preclinical model of metabolic liver disease and prove useful in the evaluation of novel liver cell therapies.