Liver-directed gene therapy is a promising approach to the treatment of human inborn errors of metabolism (IEM) due to specific liver enzyme deficiencies. The goal of this project is to develop a safe, effective liver- directed gene transfer technique for IEM by treating phenylalanine hydroxylase (PAH)-deficient Pahenu2 mice, a model of human phenylketonuria (PKU). We have recently witnessed complete correction of serum phenylalanine levels in Pahenu2 mice treated with a novel recombinant adeno-associated virus serotype 2 vector pseudotyped with serotype 8 capsid (rAAV2/8). We plan to extend this observation to evaluate long- term stability and incidence of adverse effects following rAAV2/8 administration and to further our understanding of the physiologic factors that govern phenylalanine clearance. Our specific hypothesis is that complete correction of hyperphenylalaninemia and its attendant phenotypic features will require permanent restoration of liver PAH activity in at least 10% of Pahenu2 hepatocytes. In the first aim of the project, we will investigate the physiologic thresholds that govern phenylalanine clearance in murine liver. We will transplant primary hepatocytes under a selective growth advantage into PAH deficient mice and will accurately determine the number of PAH positive hepatocytes and absolute amount of PAH enzyme activity required to correct hyperphenylalaninemia. Transplantation of hepatocytes that are either wild type and therefore express 100% normal PAH activity or are heterozygous for the Pahenu2 mutation and have <100% PAH activity will allow us to determine the interrelationship between liver PAH activity, the absolute number of PAH-expressing cells and total phenylalanine clearance. In the second aim, we will fully evaluate the efficacy and safety of rAAV2/8-mediated, liver-directed gene transfer as therapy for murine PKU. We will evaluate transduction frequency, the amount and duration of therapeutic gene expression, the effect of therapy upon physical and biochemical phenotypes of the mice, the frequency of vector integration, and the incidence of adverse effects in the animals following treatment with rAAV2/8. Our ultimate goal is to develop an effective liver-directed gene transfer method for the treatment of murine PKU. We propose that lessons learned from the treatment of murine PKU will ultimately be applicable to the treatment of human PKU and other allied IEM.
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