Purpose To investigate the utility of Magnetic Resonance Elastography (MRE)-derived mechanical properties in discriminating hepatic inflammation and fibrosis in the early stages of chronic liver diseases. Material and Methods All studies were approved by our institutional animal care and use committee. A total of 187 animals were studied, including 182 mice and 5 pigs that represent five different liver diseases with a varying combination and extent of hepatic inflammation, fibrosis, congestion and portal hypertension. We performed multifrequency 3-D MRE with shear stiffness, storage and loss modulus, and damping ratio calculated in all animal subjects. Necroinflammation, fibrosis, and portal pressure were either histologically scored or bio-chemically/physically quantified in all studied animals. Two-sided Welch's t-tests were used to evaluate the mean differences between diseased and control/sham groups. Spearman's correlation analyses were used to evaluate the relationships between the mechanical parameters and the quantitative fibrosis extent (hydroxyproline concentration) and portal pressure. Results Liver shear stiffness and storage modulus increased with progressively developed fibrosis and portal hypertension (stiffness @80Hz, 48-week-feeding, steatohepatitis/controls, 0.51±0.12/0.29±0.01kPa, p=0.02). Damping ratio and loss modulus can distinguish inflammation from fibrosis at early stages, even before the development of histologically detectable necroinflammation and fibrosis (damping ratio @80Hz, 20-week-feeding, steatohepatitis/controls, 0.044±0.012 vs. 0.014±0.008, p<0.001). Damping ratio and shear stiffness vary differently with respect to etiology of portal hypertension (i.e., congestion or cirrhosis induced). These differentiation abilities have frequency-dependent variations. Conclusion Liver stiffness and damping ratio measurements can extend hepatic MRE to potentially assess necroinflammatory, congestive, and fibrotic processes of chronic liver diseases.

We tested the hypothesis that ex vivo hepatocyte gene therapy can correct the metabolic disorder in fumarylacetoacetate hydrolase–deficient (Fah−/−) pigs, a large animal model of hereditary tyrosinemia type 1 (HT1). Recipient Fah−/−pigs underwent partial liver resection and hepatocyte isolation by collagenase digestion. Hepatocytes were transduced with one or both of the lentiviral vectors expressing the therapeutic Fah and the reporter sodium-iodide sym-porter (Nis) genes under control of the thyroxine-binding globulin promoter. Pigs received autologous transplants of hepatocytes by portal vein infusion. After transplantation, the protective drug 2-(2-nitro-4-trifluoromethylbenzyol)-1,3 cyclohexanedione (NTBC) was withheld from recipient pigs to provide a selective advantage for expansion of corrected FAH+ cells. Proliferation of transplanted cells, assessed by both immunohistochemistry and noninvasive positron emission tomography imaging of NIS-labeled cells, demonstrated near-complete liver repopulation by gene-corrected cells. Tyrosine and succinylacetone levels improved to within normal range, demonstrating complete correction of tyrosine metabolism. In addition, repopulation of the Fah−/− liver with transplanted cells inhibited the onset of severe fibrosis, a characteristic of nontransplanted Fah−/− pigs. This study demonstrates correction of disease in a pig model of metabolic liver disease by ex vivo gene therapy. To date, ex vivo gene therapy has only been successful in small animal models. We conclude that further exploration of ex vivo hepatocyte genetic correction is warranted for clinical use.

Background & Aims The neuroprotective effect of the spheroid reservoir bioartificial liver (SRBAL) was evaluated in a porcine model of drug-overdose acute liver failure (ALF). Methods Healthy pigs were randomized into three groups (standard therapy (ST) alone, ST + No-cell device, ST + SRBAL device) before placement of an implantable intracranial pressure (ICP) monitor and a tunneled central venous catheter. One week later, pigs received bolus infusion of the hepatotoxin D-galactosamine and were followed for up to 90 hours. Results At 48 hours, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90 hours after drug infusion. Pigs treated with the SRBAL, loaded with porcine hepatocyte spheroids, had improved survival (83%, n=6) compared to ST alone (0%, n=6, p=0.003) and No-cell device therapy (17%, n=6, p=0.02). Ammonia detoxification, peak levels of serum ammonia and peak ICP, and pig survival were influenced by hepatocyte cell dose, membrane pore size and duration of SRBAL treatment. Hepatocyte spheroids remained highly functional with no decline in mean oxygen consumption from initiation to completion of treatment. Conclusions The SRBAL improved survival in an allogeneic model of drug-overdose ALF. Survival correlated with ammonia detoxification and ICP lowering indicating that hepatocyte spheroids prevented the cerebral manifestations of ALF (brain swelling, herniation, death). Further investigation of SRBAL therapy in a clinical setting is warranted.

Orthotopic liver transplantation remains the only curative therapy for inborn errors of metabolism. Given the tremendous success for primary immunodeficiencies using ex-vivo gene therapy with lentiviral vectors, there is great interest in developing similar curative therapies for metabolic liver diseases. We have previously generated a pig model of hereditary tyrosinemia type 1 (HT1), an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Using this model, we have demonstrated curative ex-vivo gene and cell therapy using a lentiviral vector to express FAH in autologous hepatocytes. To further evaluate the long-term clinical outcomes of this therapeutic approach, we continued to monitor one of these pigs over the course of three years. The animal continued to thrive off the protective drug NTBC, gaining weight appropriately, and maintaining sexual fecundity for the course of his life. The animal was euthanized 31 months after transplantation to perform a thorough biochemical and histological analysis. Biochemically, liver enzymes and alpha-fetoprotein levels remained normal and abhorrent metabolites specific to HT1 remained corrected. Liver histology showed no evidence of tumorigenicity and Masson’s trichrome staining revealed minimal fibrosis and no evidence of cirrhosis. FAH-immunohistochemistry revealed complete repopulation of the liver by transplanted FAH-positive cells. A complete histopathological report on other organs, including kidney, revealed no abnormalities. This study is the first to demonstrate long-term safety and efficacy of hepatocyte-directed gene therapy in a large animal model. We conclude that hepatocyte-directed ex-vivo gene therapy is a rational choice for further exploration as an alternative therapeutic approach to whole organ transplantation for metabolic liver disease, including HT1.

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