利用携带多药耐药蛋白3（MDR3）编码序列（AAV8-MDR3）的重组腺相关病毒（rAAV）载体进行基因治疗，是治疗儿童早期进展性家族性肝内胆汁淤积症3型（PFIC3）的潜在治愈疗法。然而，PFIC3最严重的患者应在检测后尽早接受治疗，以防止不可逆的肝纤维化最终导致肝移植或死亡。这对基于rAAV的基因治疗构成了挑战，因为随着肝细胞分裂，rAAV基因组的丢失预计会降低治疗效果，并且AAV特异性中和抗体的形成排除了再次给药。在这里，我们测试了在小儿PFIC3小鼠中重新给药的策略，同时仔细评估了其致癌性 - 这是围绕rAAV治疗的特别关注点。在2周龄时，共同给予依托拉孟耐受性纳米颗粒（ImmTOR）和A8-MDR3，2周后再次给予AAV8-MDR3。8个月后，评估长期治疗效果和安全性，特别注意rAAV治疗的潜在致癌性。与ImmTOR的共同给药减轻了rAAV特异性中和抗体的形成，并启用了AAV8-MDR3的有效第二次给药，导致疾病表型的稳定校正，包括胆汁磷脂含量的恢复和肝功能的健康状态，以及防止肝纤维化，肝脾肿大和胆囊结石。此外，有效的重复rAAV给药防止了在高度易于发展肝细胞癌的动物模型中出现肝细胞癌。这些结果为通过与ImmTOR的共同给药进行rAAV再次给药提供了有力的证据，因为它导致了儿科肝代谢障碍的长期治疗效果，包括预防肿瘤发生。对于固有性肝胆疾病的基因治疗的再次给药可能是必要的，因为在肝细胞分裂和更新期间效果会减退，特别是在儿童患者中，但这种方法可能带来长期肝癌风险。携带治疗基因的病毒载体在幼年小鼠中产生了持久的进展性家族性肝内胆汁淤积症3型治愈效果，并仅在第二次给药后降低了肝癌的风险。 ©2023作者。

Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity - a particular concern surrounding rAAV treatment.AAV8-MDR3 was re-administered to infant Abcb4 -/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment.Co-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma.These outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis.Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.© 2023 The Author(s).