A mMono-Bac1.0 system for recombinant adeno-associated virus vectors with higher flexibility, throughput and yield

Recombinant adeno-associated virus (rAAV) holds immense promise for therapeutic applications, but high production costs limit its widespread clinical use, underscoring the urgent need for manufacturing breakthroughs. Here, we establish a modular Mono-Bac system with the p24 -NanoLuciferase (Nluc) reporter Bacmid for rAAV production, named mMono-Bac1.0, enhancing manufacturing flexibility, throughput and yield simultaneously. The system introduces two key technology to the pFD/Cap-(ITR-GOI)-Rep transfer vector-based Mono-Bac framework. First, a modular pGB-AAV Donor construction strategy enables one-step all-in-one AAV transfer vector assembly and effortless switching of capsids or GOIs, thus facilitating high-throughput combinatorial production. Second, an engineered p24 -Nluc Bacmid enables rapid (<5 min) quantification of infectious recombinant baculovirus (rBV) titers and real-time monitoring of Nluc activity in culture supernatants to determine the optimal harvest point (log RLU ~ 6.5), thereby maximizing rAAV yield. This system has the potential to advance baculovirus expression vector (BEV)-based rAAV manufacturing into a data-driven era, supporting efficient industrial-scale production.