Liver gene therapy for PKU using minicirde-based naked-DNA vectors

We have previously reported long-term correction of hyperphenylalaninemia and hypopigmentation of the PKU mouse model, C57BI/6-Pahenu2, after liver-directed gene transfer with recombinant adeno-associated viral (AAV) vectors. However, questions of expression stability, treatment toxicity, potential for insertional mutagenesis, and safety required for targeting newborn and paediatric patients for potential life-long treatment remain a risk for viral vector-dependent approaches. To overcome these drawbacks, we are developing non-viral gene transfer methods for liver targeting. Here we report the successful use of minicircle (MC) technology to treat murine PKU. Our MC-DNA vectors contain a de novo designed liver-specific hybrid promoter-enhancer fragment, the mouse phenylalanine hydroxylase (mini-) gene based on the endogenous cDNA (mPah), or a codon-optimzed and intron-containing version (mcoPah), plus the bovine growth hormone polyA signal. Delivery of MC vectors was mediated by hydrodynamic tail vein (HTV) injection as a liver-targeted approach. MC-Vector titration studies showed that the blood phenylalanine levels were normalized in a dose dependent manner concomitant with reversion of hypopigmentation at optimal MC-DNA concentrations for up to several months without reapplication (ongoing experiment). Upon scarifying treated PKU mice, sustained transgene expression was confirmed by the presence of hepatic PAH enzyme activity. In conclusion, MC technology offers a better safety profile and has the potential for genetherapeutic treatment of liver diseases.