Fumarylacetoacetate Hydrolase Knock-out Rabbit Model for Hereditary Tyrosinemia Type 1

Abstract

Hereditary tyrosinemia type 1 (HT1) is a severe human autosomal recessive disorder caused by the deficiency of fumarylacetoacetate hydroxylase (FAH), an enzyme catalyzing the last step in the tyrosine degradation pathway. Lack of FAH causes accumulation of toxic metabolites (fumarylacetoacetate and succinylacetone) in blood and tissues, ultimately resulting in severe liver and kidney damage with onset that ranges from infancy to adolescence. This tissue damage is lethal but can be controlled by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), which inhibits tyrosine catabolism upstream of the generation of fumarylacetoacetate and succinylacetone. Notably, in animals lacking FAH, transient withdrawal of NTBC can be used to induce liver damage and a concomitant regenerative response that stimulates the growth of healthy hepatocytes. Among other things, this model has raised tremendous interest for the in vivo expansion of human primary hepatocytes inside these animals and for exploring experimental gene therapy and cell-based therapies. Here, we report the generation of FAH knock-out rabbits via pronuclear stage embryo microinjection of transcription activator-like effector nucleases. FAH−/− rabbits exhibit phenotypic features of HT1 including liver and kidney abnormalities but additionally develop frequent ocular manifestations likely caused by local accumulation of tyrosine upon NTBC administration. We also show that allogeneic transplantation of wild-type rabbit primary hepatocytes into FAH−/− rabbits enables highly efficient liver repopulation and prevents liver insufficiency and death. Because of significant advantages over rodents and their ease of breeding, maintenance, and manipulation compared with larger animals including pigs, FAH−/− rabbits are an attractive alternative for modeling the consequences of HT1.

Endogenous toxic metabolites and implications in cancer therapy

“…As another example, 4-methylthio-2-oxobutanoate, an intermediate in the methionine salvage pathway, has been shown to effectively induce apoptosis, and the underlying mechanism for this effect is unclear [104,105]. Fumarylacetoacetate, a metabolite in the tyrosine degradation pathway that is thought to be the pathogenic metabolite in hereditary tyrosinemia [106,107], has been shown to be both mutagenic and cytotoxic, eliciting deregulation of various biological processes such as glutathione depletion, ERK activation, and cytochrome c release [108,109]. While some mechanisms such as reactivity with thiols and mitotic spindle disruption have been suggested [109], how fumarylacetoacetate interacts with these biological processes is not established.…”

Development of a rabbit model of Wiskott‐Aldrich syndrome

“…Procedures of pronuclear-stage embryo microinjection and embryo transfer were performed as previously described. 32 In brief, 6-8-month-old female New Zealand White rabbits were super ovulated with 50 IU of follicle-stimulating hormone every 12 hours for 3 days, mated with male New Zealand White rabbits, and injected with 100 IU of human chorionic gonadotrophin. At 18 hours postinjection, female rabbits were euthanized, and the oviducts were flushed with 5 mL prewarmed Dulbecco's phosphate-buffered saline-bovine serum albumin for collecting pronuclear-stage embryos.…”

“…Western blot analysis was performed according to the methods previously described. 32 The primary antibody was mouse-anti rabbit WASP (B-9) (1:2000) from Santa Cruz Biotechnology, and the secondary antibody was peroxidase-conjugated goat anti-mouse IgG (1:2000) from ProteinTech. The internal reference antibody was HRP conjugated monoclonal anti-β actin (1:2000) from KangChen.…”

Principles and Applications of Rabbit Models for Atherosclerosis Research

“…The first KO rabbits were generated by the ZFN method to target the IgM gene 28 ) , and later, we along with others, successfully generated apoCIII 29 ) and CETP KO rabbits 14 ) using the same method. Although TALENs were still used to create KO rabbits 30 ) , CRISPR-Cas9 has now been become the main method for generation of KO rabbits in this field, such as apoE and LDL receptor KO rabbits 31 , 32 ) ( Table 3 ). Therefore, one can predict that in the next few years, through these novel technologies, KO rabbits will be generated and used for translational studies of cardiovascular diseases.…”