Efficient production ofFah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer

Abstract

Hereditary tyrosinemia type I (HT1) results in hepatic failure, cirrhosis, and hepatocellular carcinoma (HCC) early in childhood and is caused by deficiency in the enzyme fumarylacetoacetate hydrolase (FAH). In a novel approach we used the chimeric adeno-associated virus DJ serotype (AAV-DJ) and homologous recombination to target and disrupt the porcine Fah gene. AAV-DJ is an artificial chimeric AAV vector containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8 and 9). The AAV-DJ vector was used to deliver the knockout construct to fetal pig fibroblasts with an average knockout targeting frequencies of 5.4%. Targeted Fah-null heterozygote fibroblasts were used as nuclear donors for somatic cell nuclear transfer (SCNT) to porcine oocytes, and multiple viable Fah-null heterozygote pigs were generated. Fah-null heterozygotes were phenotypically normal, but had decreased Fah transcriptional and enzymatic activity compared to wild-type animals. Conclusion This study is the first to use a recombinant chimeric AAV vector to knockout a gene in porcine fibroblasts for the purpose of SCNT. In using the AAV-DJ vector we observed targeting frequencies that were higher than previously reported with other naturally occurring serotypes. We expect that the subsequent generation of FAH-null homozygote pigs will serve as a significant advancement for translational research in the areas of metabolic liver disease, cirrhosis and HCC.

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

“…13,14 The acute phenotype of HT1 in FAH À/À pigs, seen after withdrawal of NTBC, closely resembles acute-onset HT1 in humans, which is characterized by acute liver failure and death. 14,22 In humans, treatment with NTBC abolishes the acute complications of HT1 in most patients.…”

“…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.…”

“…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.…”

Genetically modified pigs for biomedical research

“…The underlying cause for fetus-to-fetus or fibroblast variability in gene targeting efficiency remains unknown. Recently, another study used a chimeric rAAV vector (rAAV-DJ), containing hybrid capsid sequences from three naturally occurring serotypes (AAV2, 8, and 9), and SCNT to successfully generate cloned pigs carrying targeted deletions in the fumarylacetoacetate hydrolase (FAH) gene as a model for studying human liver diseases like cirrhosis and hepatocellular carcinoma (Hickey et al 2011). Similar efficiency and variability of gene targeting efficiency (2.3 % in male fibroblasts and 8.5 % in female fibroblasts) were also observed in this study much like in the study by Rogers et al (2008b).…”