Stable transduction of the neonatal mouse liver using a hybrid rAAV/sleeping beauty transposon gene delivery system

Abstract Background Conventional adeno‐associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/ piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty , has been widely used for ex vivo gene delivery; however liver‐targeted delivery using a hybrid rAAV/ Sleeping Beauty approach remains relatively unexplored. Methods We investigated the capacity of a Sleeping Beauty (SB)‐based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC). Results At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4‐ to 8‐fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC‐deficient Spf ash mouse following elimination of residual endogenous murine OTC. Conclusions The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long‐term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.