Development of an inducible stable producer cell line for rAAV production.

Adeno-associated virus (AAV) is a widely preferred in vivo gene therapy vector for clinical trials due to its numerous advantages. However, manufacturing recombinant AAV (rAAV) using transient transfection methods poses significant challenges. Stable cell lines present a promising alternative for large-scale, continuous production, addressing the limitations of transient transfection and enabling more efficient rAAV manufacturing. In this study, we developed an inducible stable producer cell line by integrating transfer (GFP-containing) and assembly (Cap-containing) cassettes into a previously established inducible stable packaging cell line using transposon-mediated integration. The stable cell pool was initially evaluated for genome copy number of all viral components and rAAV production. Detection of genome titer signals in the pool confirmed successful integration, leading to the isolation of single clones. These clones were further screened for GFP and Cap genome copy numbers, with those exhibiting comparable or higher copy numbers than the stable pool undergoing comprehensive genome copy analysis for all viral components. The top-performing clone was identified and characterized in detail. Following doxycycline induction, genome titer (3.73E11 vg/L) and capsid titer (2.01E11 cp/L) were detected, confirming the successful establishment of the inducible stable producer cell line. This study highlights the feasibility of developing inducible stable cell lines for rAAV production through refactored viral vector components and a combination of site-specific and transposon-mediated integration approaches.