Correction in female PKU mice by repeated administration of mPAH cDNA using phiBT1 integration system

Abstract

Phenylketonuria (PKU) is a metabolic disorder secondary to a hepatic deficiency of phenylalanine hydroxylase (PAH) that predisposes affected children to develop severe and irreversible mental retardation. We have previously reported the complete and permanent correction of the hyperphenylalaninemic and hypopigmentation phenotypes in male, but not female, PKU mice after genome-targeted delivery of murine PAH (mPAH) complementary DNA (cDNA) in a phiBT1 bacteriophage integration system. Here we show that sequential administration of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing cassettes in the phiBT1 integration system led to distinct and non-overlapping populations of green and red fluorescent hepatocytes in vivo. The hyperphenylalaninemic and hypopigmentation phenotypes of female PKU mice were completely corrected after 10 weekly administrations of mPAH cDNA. Importantly, there was no apparent liver pathology in mice even after 10 consecutive administrations of the phiBT1 integration system. The results indicate that repeated administration of transgenes in the phiBT1 integration system can lead to their genome-targeted integration in a diverse population of hepatocytes and result in the elevation of transgene expression levels in a cumulative manner, which can be utilized to overcome insufficient transgene expression owing to low genome integration frequencies in a gene therapy paradigm for metabolic disorders.