Comparative Transcriptomic Profiling Reveals Differences in Initiation of Antiviral Response in Low rAAV Producing...

Recombinant adeno-associated virus (rAAV) vectors have become the leading platform for in vivo gene delivery, yet their industrial-scale production remains inefficient and costly. Current manufacturing, which primarily relies on transient triple-plasmid transfection in HEK293-derived cells, is limited by low vector yields, high empty-to-full capsid ratios, and pronounced batch-to-batch variability. To contribute to the understanding of underlying host factors, we compared transcriptomic profiles of five HEK293-derived producer lines with varying rAAV yields over a 72-hour production time course. The low-producing BalCD line showed poor transfection efficiency, reduced plasmid-derived transcript levels, exclusive interferon expression and a rapid activation of interferon-stimulated genes (ISG), indicating strong antiviral restriction for vector productivity. In contrast, high-producing lines mounted a reduced inflammatory and innate immune response with limited induction of antiviral genes, supporting sustained vector production. Expression patterns of high-producing cell lines further exhibited transcriptional signatures resembling those seen for producer cells arrested in the G0/G1 cell cycle phase, which potentially favors efficient vector genome replication and capsid assembly. The observed differences in the regulation of antiviral and cell cycle-associated pathways highlight key molecular determinants of rAAV productivity and provide a foundation for targeted host engineering strategies aimed at enhancing rAAV manufacturing efficiency.