S. Angarita1, B. Truong1, M. Lin1, S. Khoja1, A. Lam1, S. Duarte1, G. S. Lipshutz1 1David Geffen School Of Medicine,Surgery,Los Angeles, CALIFORNIA, USA
Introduction: Hepatocytes perform a variety of functions including protein synthesis, detoxification, and biliary excretion. Inherited abnormalities of any one of the multitude of enzymes in the liver can lead to a metabolic disorder. Liver transplantation (LT) can cure patients with liver-based metabolic disorders. However, LT is dependent on the availability of donor livers which are particularly limited for infants and neonates due to organ scarcity and size limitations. We are presently exploring the transplantation of isolated primary hepatocytes as a minimally invasive alternative to LT. In these studies, we demonstrate that human hepatocytes can be used to treat arginase deficiency, an inherited disorder of nitrogen metabolism typically presenting in the first two years of life, by hepatocyte repopulation of the liver in a murine model of the disorder.
Methods: Conditional arginase deficient mice were mated with a mouse model deficient in fumarylacetoacetate hydrolase (FAH) and with Rag2 and IL2-Rγ mutations. The FAH knockout results in a defect in the tyrosine catabolic pathway, resulting in highly reactive toxic metabolites that cause hepatocellular injury. Toxicity can be prevented by oral administration of 2-(2-nitro-4-trifluoro-methylbenzoyl)-1,3-cyclohexanedione (NTBC), which blocks the tyrosine pathway upstream. This enzymatic defect provides a selection advantage for transplanted cells. On day -1, a uroplasminogen-expressing adenoviral vector was administered IV. On day 0, human hepatocytes were isolated and 1×106 were administered by intrasplenic injection (n=12). As the initial number of administered hepatocytes was too low to prevent hepatotoxicity-induced mortality, NTBC was added to the drinking water of the mice at regular intervals with eventual withdrawal for 3 weeks at a time. Ninety days after hepatocyte transplantation, 2×1011 genome copies of AAV-TBG-Cre recombinase was administered IV to knock out endogenous arginase expression in hepatocyte-transplanted mice and controls. Survival was followed; amino acids and ammonia were studied 30 days later.
Results: All control mice (n=16) died by day 34 (26.2 ± 3.4) while all except one human hepatocyte transplanted mice survived. Ammonia and amino acids were analyzed in both groups comparing day 60 (30 days before arginase knockout) and day 150 (30 days after AAV-TBG-Cre administration). Ammonia, arginine and glutamine (elevated in urea cycle dysfunction) were well-controlled in the transplanted group, while in the controls they were markedly abnormal.
Conclusion: These studies demonstrate that human hepatocyte repopulation in the murine liver can result in cure of arginase deficiency, and likely is also applicable to other metabolic disorders that are currently treated by LT. With success of these proof of principal studies, we are presently exploring methods that may allow for selective advantage of transplanted hepatocytes in humans with such disorders.