Host determinants of exo-rAAV production revealed by extended gene expression analysis in HEK293 cells.

Recombinant adeno-associated viruses (rAAVs) are leading vectors for gene therapy, yet their large-scale manufacturing remains constrained by low scalability and high costs. Here, we present an integrated systems biology and process engineering strategy to intensify rAAV production through the extended gene expression (EGE) methodology. EGE has been reported to significantly increase rAAV yields, enhanced viral egress, and promote the secretion of exosome-associated AAVs (exo-AAVs), resulting in higher extracellular titers and improved vector potency. Quantitative multiplexed proteomics revealed coordinated remodeling of host-cell pathways involved in vesicle trafficking, lipid biosynthesis, protein homeostasis, and nuclear transport. Functional validation identified key host regulators of exo-rAAV biogenesis and secretion, including CD63, VPS37B, SMPD3, and SNAP47. Targeted modulation of these pathways enabled rational enhancement of extracellular rAAV recovery in standard batch processes. Using a DoE-based mixture design, we achieved 50% viral egress into the supernatant, with >21% being exo-AAVs, demonstrating efficient translation of the EGE phenotype into scalable batch manufacturing. Collectively, this work provides a scalable and cost-effective alternative production platform with the advantages of the EGE methodology. These findings can help advance next-generation rAAV manufacturing and support the development of gene therapy vectors.