Abstract
Recent advances in three-dimensional (3D) culture platforms have enabled organoids to serve as physiologically relevant models for recapitulating human biology and assessing therapeutic efficacy and toxicity. Despite their promise, their complex architecture presents significant challenges for efficient gene delivery, thereby limiting their broader application in drug discovery and translational research. To overcome this challenge, we developed a sequential treatment strategy that combines polybrene (PB), which facilitates viral entry, and hydroxychloroquine (HCQ), which modulates endosomal processing. By applying PB as a pre-treatment and HCQ as a post-treatment, we achieved an approximate 1.3- to 2-fold increase in adeno-associated virus (AAV) transduction efficiency in both retinal and liver organoid models compared to single-agent treatments, and a 1.7- to 2.5-fold increases compared to treatment with virus alone. Importantly, this combinatorial treatment preserved cellular integrity, as confirmed by minimal TUNEL assay and high overall viability. Our findings demonstrate that sequential administration of PB and HCQ significantly improves AAV transduction in 3D retinal and liver organoid systems, offering a robust method to improve gene delivery. This approach not only overcomes current limitations in organoid-based research but also supports the development of more predictive platforms for evaluating AAV vectors and advancing gene therapy applications.