Comparative Transcriptomic Profiling Reveals Differences in Initiation of Antiviral Response in Low rAAV Producing HEK293 Suspension Cells

Abstract

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.