Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system

 Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV)shows promising therapeutic efficacy in animal models and adult-focused clinical trials.This promise, however, is not directly translatable to the growing liver, where high ratesof hepatocellular proliferation are accompanied by loss of episomal rAAV genomes andsubsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBactransposon vector system combining the highly efficient liver-targeting properties ofrAAV with stable piggyBac-mediated transposition of the transgene into the hepatocytegenome. Transposition efficiency was first tested using an enhanced green fluorescentprotein expression cassette following delivery to newborn wild-type mice, with a 20-foldincrease in stably gene-modified hepatocytes observed 4 weeks posttreatment comparedto traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal periodwas sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase–deficient Spfash mouse and the neonatal lethal argininosuccinate synthetaseknockout mouse. Finally, transposon integration patterns were analyzed, revealing127,386 unique integration sites which conformed to previously published piggyBacdata. Conclusion: Using a hybrid rAAV/piggyBac transposon vector system, we achievedstable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defectscould be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, andengineered refinements in the safety profile of piggyBac transposase-mediated integration.