176. An Inhibitor of Fumarylacetoacetate Hydrolase (Fah) for Selection of Transplanted Hepatocytes and Gene Correction In Vivo

Abstract

Mice deficient in the tyrosine catabolic enzyme Fah are an excellent model for liver repopulation by selection. Fah positive cells from several different origins can engraft and correct the phenotype of a Fah-mutant liver. Non-cell based gene therapy leading to the in situ correction of the Fah-deficiency also works and repopulation levels of >90% are routinely achieved. Thus, hepatic Fah deficiency provides a powerful in vivo selection environment for cells genetically resistant to the enzyme deficiency. In order to mimic the same selective pressure in animals not genetically deficient in Fah, we developed a small molecule inhibitor of Fah. 4-[(2-carboxyethyl)- hydroxyphosphinyl]-3-oxobutyrate (CEHPOBA) has a nanomolar Ki for Fah and was injected intraperitoneally into C57/BL6 mice at a concentration of 1|[mu]|moles per gram body weight. In a first round of experiments, the duration of Fah inhibition in vivo was tested. We found that a once-daily injection resulted in a lasting enzyme repression. Next, we tested if transplanted hepatocytes could be selected under continuous CEHPOBA administration. As shown previously, hepatocytes mutant for the homogentisic acid dioxygenase (Hgd) an enzyme upstream of Fah in tyrosine catabolism are able to repopulate the livers of mice with Fah-deficiency. We therefore transplanted 500,000 sex-mismatched Hgd-mutant hepatocytes into Fah+ C57/BL6 mice and concomitantly started CEHPOBA treatment. Treated animals appeared healthy and displayed no major side-effects. After 4 weeks of daily CEHPOBA injection, the livers were harvested and analyzed by semi-quantitative PCR and histology for the presence of Y-chromosome. 1|[ndash]|10% of total hepatocytes were Y-chromosome positive. Transplanted, Y-chromosome positive hepatocytes displaying Fah wild type formed nodules proving clonal expansion and ongoing selection of the genetically favored hepatocytes. Our results show that a significant selection of genetically resistant hepatocytes can be achieved by CEHPOBA administration in vivo. This proof-of-principle opens the door to a whole new field of experimental and therapeutic options for cell transplantation and gene correction of liver diseases in vivo. CEHPOBA resistance occurs in cells lacking enzymes upstream of Fah in tyrosine metabolism and could therefore be achieved by shRNAs targeting these genes. This strategy is currently under investigation.