Enhanced Adeno‐Associated Virus Production in HEK293 Cells via Dual Inducible Control of Cap and Rep Proteins

ABSTRACT Adeno‐associated viruses (AAVs) are promising vectors for gene therapy due to their safety, stability, and broad tissue tropism. However, current AAV production strategies face challenges related to low yield and poor quality, limiting their ability to meet clinical demands. In this study, we developed a novel Cap‐Rep dual control system for AAV production that delays Cap expression via mifepristone‐dependent promoter and suppresses Rep78 expression at late stage using a tetracycline‐dependent promoter. By delaying Cap protein expression, synchronization between viral DNA replication and Cap expression was achieved, leading to increased viral yield. Additionally, suppressing Rep78 protein expression at late stage increased cell density and viability further boosting AAV production. This novel Cap‐Rep dual control system increased AAV5 yields from 8.56 × 10 10 to 3.14 × 10 11 vg/mL (3.67‐fold) and full‐to‐empty ratio by 1.54‐fold. Furthermore, this system enhanced production of AAV1 (3.94‐fold), AAV2 (4.71‐fold), AAV6 (4.05‐fold), and AAV9 (2.13‐fold) production while transduction efficiency is maintained. Our findings demonstrate that this novel Cap‐Rep dual control system significantly enhances AAV manufacturing efficiency, providing a promising solution to reduce the production cost of AAV vectors.