Primary hyperoxaluria type I (PH1) is an autosomal recessive disease caused by excessive oxalate production by hepatocytes due to peroxisomal alanine-glyoxylate aminotransferase (AGT) deficiency, leading to increased conversion of glyoxylate to oxalate. Increased urinary oxalate excretion causes nephrocalcinosis and urolithiasis, leading to renal failure and consequent tissue oxalosis with life-threatening complications. Combined liver-kidney transplantation, the definitive treatment of PH1, is not always successful because of large body oxalate stores. Hepatocyte transplantation, which is much less invasive, could be used potentially as a preemptive treatment. As hepatocytes overproduce oxalate in PH1, a significant fraction of mutant host hepatocytes must be replaced by AGT-competent cells, which is beyond the capacity of currently used hepatocyte transplantation procedures. Also, transplantation of allogeneic hepatocytes requires immunosuppression to prevent graft rejection. Three recent developments in our laboratory offer potential solutions to these problems. (a) We have generated a new Agxt-1 gene-deleted mouse model of PH1 (Agxt-/-) and a new transgenic mouse expressing a mutant human AGXT, which is most common in PH1 patients, in the Agxt-null background. Second, we have devised strategies to massively repopulate the liver by providing proliferative advantage to transplanted hepatocytes, using preparative hepatic X-irradiation (HIR) and expressing hepatocyte growth factor (HGF) as a mitotic stimulant. Third, we found that expressing adenoviral E3 genes or down-regulating Fas in donor hepatocytes prevents their allograft rejection, without suppressing the host immune system. Specific Aim 1 is to determine the minimum effective HIR dose and the optimum temporal relationship between HIR, HGF expression and hepatocyte transplantation using Agxt-/- recipients and congeneic LacZ- transgenic donor mice. We will determine the level of hepatic repopulation needed for therapeutically significant amelioration of hyperoxaluria, and preventing nephrocalcinosis and urolithiasis in the face of oral ethylene glycol challenge or unilateral nephrectomy. We will identify cell cycle regulator proteins that are required for HIR-based hepatic repopulation. In Specific Aim 2 we will evaluate two approaches to abrogate allograft rejection: (a) Autologous primary hepatocytes isolated from a resected liver lobe of Agxt- /- mice will be transduced with human or mouse AGXT using lentiviral vectors, and then transplanted back into the donor to avoid allorejection. (b) Primary hepatocytes isolated from allogeneic wildtype donors will be transduced ex vivo with AdE3 genes or shRNA against Fas, before transplantation into Agxt-/- recipients. Successful completion of these studies will represent a major step toward hepatocyte-based cure of a large number of liver-based inherited metabolic disorders, including PH1. PUBLIC HEALTH RELEVANCE: This research project is aimed at developing novel therapies for primary hyperoxaluria-1 based on extensive repopulation of the liver with transplanted hepatocytes, and designing strategies for circumventing allograft rejection of hepatocytes.