Fumarylacetoacetate Hydrolase-deficient Mice Research Articles

A Novel Humanized Model of NASH and Its Treatment With META4, A Potent Agonist of MET.

Background & AimsNonalcoholic fatty liver disease is a frequent cause of hepatic dysfunction and is now a global epidemic. This ailment can progress to an advanced form called nonalcoholic steatohepatitis (NASH) and end-stage liver disease. Currently, the molecular basis of NASH pathogenesis is poorly understood, and no effective therapies exist to treat NASH. These shortcomings are due to the paucity of experimental NASH models directly relevant to humans.MethodsWe used chimeric mice with humanized liver to investigate nonalcoholic fatty liver disease in a relevant model. We carried out histologic, biochemical, and molecular approaches including RNA-Seq. For comparison, we used side-by-side human NASH samples.ResultsHerein, we describe a “humanized” model of NASH using transplantation of human hepatocytes into fumarylacetoacetate hydrolase-deficient mice. Once fed a high-fat diet, these mice develop NAFLD faithfully, recapitulating human NASH at the histologic, cellular, biochemical, and molecular levels. Our RNA-Seq analyses uncovered that a variety of important signaling pathways that govern liver homeostasis are profoundly deregulated in both humanized and human NASH livers. Notably, we made the novel discovery that hepatocyte growth factor (HGF) function is compromised in human and humanized NASH at several levels including a significant increase in the expression of the HGF antagonists known as NK1/NK2 and marked decrease in HGF activator. Based on these observations, we generated a potent, human-specific, and stable agonist of human MET that we have named META4 (Metaphor) and used it in the humanized NASH model to restore HGF function.ConclusionsOur studies revealed that the humanized NASH model recapitulates human NASH and uncovered that HGF-MET function is impaired in this disease. We show that restoring HGF-MET function by META4 therapy ameliorates NASH and reinstates normal liver function in the humanized NASH model. Our results show that the HGF-MET signaling pathway is a dominant regulator of hepatic homeostasis.

A High-Content Screen Identifies MicroRNAs That Regulate Liver Repopulation After Injury in Mice

Liver regeneration is impaired in mice with hepatocyte-specific deficiencies in microRNA (miRNA) processing, but it is not clear which miRNAs regulate this process. We developed a high-throughput screen to identify miRNAs that regulate hepatocyte repopulation after toxic liver injury using fumarylacetoacetate hydrolase-deficient mice. We constructed plasmid pools encoding more than 30,000 tough decoy miRNA inhibitors (hairpin nucleic acids designed to specifically inhibit interactions between miRNAs and their targets) to target hepatocyte miRNAs in a pairwise manner. The plasmid libraries were delivered to hepatocytes in fumarylacetoacetate hydrolase-deficient mice at the time of liver injury via hydrodynamic tail-vein injection. Integrated transgene-containing transposons were quantified after liver repopulation via high-throughput sequencing. Changes in polysome-bound transcripts after miRNA inhibition were determined using translating ribosome affinity purification followed by high-throughput sequencing. Analyses of tough decoy abundance in hepatocyte genomic DNA and input plasmid pools identified several thousand miRNA inhibitors that were significantly depleted or increased after repopulation. We classified a subset of miRNA binding sites as those that have strong effects on liver repopulation, implicating the targeted hepatocyte miRNAs as regulators of this process. We then generated a high-content map of pairwise interactions between 171 miRNA-binding sites and identified synergistic and redundant effects. We developed a screen to identify miRNAs that regulate liver repopulation after injury in live mice.

Pericentral hepatocytes produce insulin-like growth factor-2 to promote liver regeneration during selected injuries in mice.

IGF-2 is produced by pericentral hepatocytes to promote hepatocyte proliferation and repair tissue damage in the setting of chronic liver injury, which is distinct from the signaling that occurs post-PHx. (Hepatology 2017;66:2002-2015).

Therapeutic Liver Reconstitution With Murine Cells Isolated Long After Death

Due to the shortage of donor organs, many patients needing liver transplantation cannot receive one. For some liver diseases, hepatocyte transplantation could be a viable alternative, but donor cells currently are procured from the same sources as whole organs, and thus the supply is severely limited. Here, we investigated the possibility of isolating viable hepatocytes for liver cell therapy from the plentiful source of morgue cadavers. To determine the utility of this approach, cells were isolated from the livers of non-heart-beating cadaveric mice long after death and transplanted into fumarylacetoacetate hydrolase-deficient mice, a model for the human metabolic liver disease hereditary tyrosinemia type I and a stringent in vivo model for hepatic cell transplantation. Surprisingly, complete and therapeutic liver repopulation could be achieved with hepatocytes derived up to 27 hours post mortem. Competitive repopulation experiments showed that cadaveric liver cells had a repopulation capacity similar to freshly isolated hepatocytes. Importantly, viable hepatocytes also could be isolated from cadaveric primate liver (monkey and human) efficiently. These data provide evidence that non-heart-beating donors could be a suitable source of hepatocytes for much longer time periods than previously thought possible.

405. Correction of the Murine Model of Hereditary Tyrosinemia Type I Using Messenger RNA as a Source of Transposase for Sleeping Beauty Mediated Integration of the FAH Gene

Sleeping Beauty (SB) is a DNA transposon capable of mediating chromosomal integration and stable expression in vertebrate cells when co-delivered with a source of transposase. In all pre-clinical reports where SB-mediated gene insertion in somatic cells has been used to correct mouse models of human disease, the transposase component has been provided as a co-delivered DNA molecule that has the potential for integration into the host cell genome. Integration and continued expression of a gene encoding SB transposase could be problematic if it led to remobilization and reintegration of transposons. Such continued expression of transposase is a key safety concern in development of the SB transposon system for clinical applications. As an alternate source of transposase, we have previously shown that in vitro transcribed transposase-encoding messenger RNA (mRNA) can effectively mediate transposon insertion both in vitro and in mouse liver (Wilber et al., Molecular Therapy, 2005, in press). Here, we test the use of transposase- encoding mRNA plus transposon DNA for gene therapy of hereditary tyrosinemia type I by first evaluating several parameters for systemic delivery and expression of mRNA in mice. We also introduce a method to quantitatively track repopulating liver cells by in vivo bioluminescence imaging after co-delivery of a DNA or RNA source of transposase with a bi-functional transposon encoding both mouse fumaryl acetoacetate hydrolase (FAH) and firefly luciferase (luc) genes in FAH deficient mice by rapid, high-volume injection into the tail vein. Liver repopulation was quantitatively monitored over time by increasing luc activity, measured as light emitted from the liver. Using this method, we determined that supplying SB transposase in the form of mRNA results in selective repopulation of corrected hepatocytes with stable co-expression of both FAH and luc. Plasma taken from animals 5 months after co-infusion with transposase mRNA contained levels of succinylacetone (the clinical determinant of tyrosinemia) that were nearly normalized. Amino acid levels were also normalized, suggesting normal liver metabolism of catabolized protein products (including urea and glucose). We further demonstrated the stability of integration by transplanting hepatocytes (250,000) into FAH deficient recipient mice. All transplanted animals survived NTBC withdrawal and gained weight consistently over a period of 90 days and demonstrated stable expression of luc. In summary, we demonstrate for the first time that transposase-encoding mRNA can be used to mediate non-viral gene therapy resulting in complete phenotypic correction that is stable and not associated with any incidents of cellular transformation.