A hybrid piggyBac AAV-transposon and LNP-transposase for treating arginase deficiency: A 100-fold dose reduction compared to AAV alone.

Arginase deficiency, a rare urea cycle disorder, causes progressive neurological impairment and spastic diplegia. Traditional adeno-associated virus (AAV) gene therapy can prevent disease in murine models but requires high doses associated with potential dose-dependent clinical toxicity and may exhibit waning durability when administered early in life. To enhance potency and persistence, we applied piggyBac-mediated transposition to integrate a functional synthetic copy of the human arginase 1 gene (hARG1) into the genome. A hybrid system was developed using AAV9 to deliver the piggyBac transposon carrying this hARG1 expression cassette, paired with a lipid nanoparticle (LNP) delivering mRNA for the super piggyBac (SPB) transposase. Adult conditional Arg1 knockout mice received the hARG1 transposon AAV and catalytically active or inactive SPB mRNA-LNPs, while a traditional non-integrating AAV served as the control. Following disease induction on day 24, all vehicle-treated mice perished by day 50. Mice treated with catalytically active hybrid vectors at doses of 5 × 1011-1 × 1013 genome copies/kg (gc/kg) survived for 9 months, maintained stable arginine, ammonia, and guanidinoacetic acid, and exhibited normalized ureagenesis. Comparable correction was only observed with traditional AAV at 5 × 1013 gc/kg-100-fold higher. These findings demonstrate that transient LNP-mediated transposase delivery combined with AAV-transposon vectors enables durable hepatic correction and markedly improves the therapeutic index for systemic gene transfer.