Following liver-targeted AAV therapy in pediatric patients, natural liver growth and hepatocyte regeneration result in a decline in AAV episome-driven expression. Thus, the development of a strategy for successful AAV readministration to restore AAV episome pools may be essential for many pediatric liver disorders. For example, progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare hepatic condition that typically presents during infancy or early childhood and results from improper transport of phosphatidylcholine from hepatocytes into the bile due to mutations in the ABCB4 gene. We have tested a readministration strategy combining a recombinant AAV8-ABCB4 (VTX-803(8)) with immunotolerogenic rapamycin-containing nanoparticles (ImmTOR) in very young mice (2 weeks old). Long-term effects on PFIC3 disease manifestations and evidence of tumorigenesis were analyzed 8 months later. Only animals given ImmTOR on the first administration and treated with a second rAAV administration achieved a long-term therapeutic effect with normalization of serum biomarkers of cholestasis, hepatosplenomegaly, biliary phospholipid content and liver fibrosis. At 8 months, control animals presented with liver lesions, gallstones (41%), and atypical hepatocellular neoplasms or dysplastic nodules detected via histopathological analysis (52%). In contrast, all mice that achieved successful transgene expression following repeat VTX-803(8) administration with ImmTOR did not show any sign of tumorigenesis. Quantification of preneoplasia markers supported these findings, including differences in hepatocyte proliferation, bile duct proliferation and incidence of DNA double strand breaks. These outcomes provide evidence to mitigate safety concerns of increased risk of hepatocellular carcinoma following AAV treatment in pediatrics or in patients with liver damage.