1097. Complete and Persistent Phenotypic Correction of Phenylketonuria in Mice by Site-Specific Genome Integration of Phenylalanine Hydorxylase cDNA

Abstract

Top of pageAbstract Phenylketonuria (PKU) is a metabolic disorder that predisposes affected children to severe and irreversible mental retardation, and is secondary to a deficiency of the hepatic enzyme phenylalanine hydroxylase (PAH), that catalyses the conversion of phenylalanine to tyrosine. In an attempt to correct this metabolic deficiency, we explored the potential of using a bacteriophage integrase system to achieve site-specific genome integration of murine PAH cDNA in the livers of Phenylketonuric mice. The integrase from Streptomyces phage |[Phi]|BT1 mediates efficient recombination between the phage attachment site attP and the bacterial attachment site attB, results in the integration of phage DNA into the bacterial genome in a site-specific manner. Here we showed that this phage enzyme functioned efficiently both in Escherichia coli and mouse cells in vitro. Several naturally occurring |[ldquo]|pseudo|[rdquo]| attP sites in the mouse genome, which contain partial sequence identity to the wild type attP site and are recognizable by the phage integrase, have been identified and molecularly characterized. In the presence of the phage integrase, naked plasmid DNA containing a wild-type attB site can be integrated specifically into these pseudo attP site permanently both in culture mouse cells and in the livers of mice. We further demonstrated that the addition of nuclear localization signal sequences to the C-terminus of the phage integrase enzyme enhanced the efficiency for transgene integration into the mouse genome. Using this phage integration system we delivered mouse PAH cDNA to the livers of PKU mice by hydrodynamic injection of plasmid DNA and showed that serum phenylalanine levels in the treated mice decreased significantly and persistently. After 3 applications, serum phenylalanine levels in all treated mice were reduced to the normal range and remained stable for more than 6 weeks. Furthermore, their fur color also changed from gray to black, indicating the reconstitution of melatonin biosynthesis as a result of available tyrosine derived from phenylalanine hydroxylation. Thus the bacterio-phage integrase represents an effective site-specific genome integration system in mammalian cells and can be of great value in plasmid DNA-mediated gene therapy of a multitude of genetic disorders.