Augmentation of transgene-encoded protein after neonatal injection of adeno-associated virus improves hepatic copy number without immune responses

BACKGROUND: Achieving persistent expression is a prerequisite for genetic therapies for inherited metabolic enzymopathies. Such disorders potentially could be treated with gene therapy shortly after birth to prevent pathology. However, rapid cell turnover leads to hepatic episomal vector loss, which diminishes effectiveness. The current studies assessed whether tolerance to transgene proteins expressed in the neonatal period is durable and if the expression may be augmented with subsequent adeno-associated virus (AAV) administration. METHODS: AAV was administered to mice on day 2 with reinjection at 14 or at 14 and 42 d with examination of changes in hepatic copies and B and T cell-Mediated immune responses. RESULTS: Immune responses to the transgene protein and AAV were absent after neonatal administration. Reinjection at 14 or at 14 and 42 d resulted in augmented expression with greater hepatic genome copies. Unlike controls, immune responses to transgene proteins were not detected in animals injected as neonates and subsequently. However, while no immune response developed after neonatal administration, anticapsid immune responses developed with further injections suggesting immunological ignorance was the initial mechanism of unresponsiveness. CONCLUSIONS: Persistence of transgene protein allows for tolerance induction permitting readministration of AAV to reestablish protein levels that decline with growth.