Autologous Gene and Cell Therapy Provides Safe and Long-Term Curative Therapy in A Large Pig Model of Hereditary Tyrosinemia Type 1.

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.

Distinguishing between Hepatic Inflammation and Fibrosis with MR Elastography.

Purpose To investigate the utility of magnetic resonance (MR) elastography-derived mechanical properties in the discrimination of hepatic inflammation and fibrosis in the early stages of chronic liver diseases. Materials and Methods All studies were approved by the institutional animal care and use committee. A total of 187 animals were studied, including 182 mice and five pigs. These animals represented five different liver diseases with a varying combination and extent of hepatic inflammation, fibrosis, congestion, and portal hypertension. Multifrequency three-dimensional MR elastography was performed, and shear stiffness, storage modulus, shear loss modulus, and damping ratio were calculated for all animals. Necroinflammation, fibrosis, and portal pressure were either histologically scored or biochemically and physically quantified in all animals. Two-sided Welch t tests were used to evaluate mean differences between disease and control groups. Spearman correlation analyses were used to evaluate the relationships between mechanical parameters and quantitative fibrosis extent (hydroxyproline concentration) and portal pressure. Results Liver stiffness and storage modulus increased with progressively developed fibrosis and portal hypertension (mean stiffness at 80 Hz and 48-week feeding, 0.51 kPa ± 0.12 in the steatohepatitis group vs 0.29 kPa ± 0.01 in the control group; P = .02). Damping ratio and shear loss modulus can be used to distinguish inflammation from fibrosis at early stages of disease, even before the development of histologically detectable necroinflammation and fibrosis (mean damping ratio at 80 Hz and 20-week feeding, 0.044 ± 0.012 in the steatohepatitis group vs 0.014 ± 0.008 in the control group; P < .001). Damping ratio and liver stiffness vary differently with respect to cause of portal hypertension (ie, congestion- or cirrhosis-induced hypertension). These differentiation abilities have frequency-dependent variations. Conclusion Liver stiffness and damping ratio measurements can extend hepatic MR elastography to potentially enable assessment of necroinflammatory, congestive, and fibrotic processes of chronic liver diseases. © RSNA, 2017 Online supplemental material is available for this article.

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

Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disease caused by deficiency in fumarylacetoacetate hydrolase, the last enzyme in the tyrosine catabolic pathway. In this study, we investigated whether fumarylacetoacetate hydrolase deficient (FAH-/-) pigs, a novel large-animal model of HT1, develop fibrosis and cirrhosis characteristic of the human disease. FAH-/- pigs were treated with the protective drug 2-(2-nitro-4-trifluoromethylbenzoyl)-1, 3 cyclohexandione (NTBC) at a dose of 1 mg/kg per day initially after birth. After 30 days, they were assigned to one of three groups based on dosing of NTBC. Group 1 received ≥0.2 mg/kg per day, group 2 cycled on/off NTBC (0.05 mg/kg per day × 1 week/0 mg/kg per day × 3 weeks), and group 3 received no NTBC thereafter. Pigs were monitored for features of liver disease. Animals in group 1 continued to have weight gain and biochemical analyses comparable to wild-type pigs. Animals in group 2 had significant cessation of weight gain, abnormal biochemical test results, and various grades of fibrosis and cirrhosis. No evidence of hepatocellular carcinoma was detected. Group 3 animals declined rapidly, with acute liver failure. In conclusion, the FAH-/- pig is a large-animal model of HT1 with clinical characteristics that resemble the human phenotype. Under conditions of low-dose NTBC, FAH-/- pigs developed liver fibrosis and portal hypertension, and thus may serve as a large-animal model of chronic liver disease.

Curative ex vivo liver-directed gene therapy in a pig model of hereditary tyrosinemia type 1.

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 symporter (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.

Pivotal preclinical trial of the spheroid reservoir bioartificial liver.

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 90h. RESULTS: At 48h, all animals had developed encephalopathy and biochemical changes confirming ALF; extracorporeal treatment was initiated and pigs were observed up to 90h 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.

Laparoscopic inguinal hernia repair--an unusual complication.

Laparoscopic inguinal hernia repair is associated with minimal postoperative pain, quicker return to normal activities, and very low recurrence rates. We describe an unusual complication after a laparoscopic total extraperitoneal repair.