Human embryonic kidney cells HEK293 are widely used in biopharmaceutical manufacturing, with a recent surge particularly in recombinant adeno‐associated virus production. Despite their industrial relevance, comprehensive data on their genomic background and stability remains limited. Here, we systematically analyze the genetic landscape of various HEK293 cell lines in response to cultivation conditions, clonal selection, genetic manipulation and over time in culture. Adherent HEK293 were adapted to suspension growth using different serum‐free media. Whole genome sequences from these cell lines were analyzed together with previously published data from additional variants in common use. All data sets were aligned against the human reference genome, enabling the assessment of genome stability by evaluation of variants and revealing a conserved genetic core across all lines, regardless of cultivation history or phenotypic divergence. Evaluation of the functional implications of conserved core mutations identified an enrichment in genes related to cellular structure, morphology and cellular connectivity. The distribution of structural variants and single nucleotide polymorphisms indicated a gradual accumulation of mutations over time in culture rather than abrupt shifts in response to environmental changes. Notably, the integrated adenoviral genes remained highly conserved with respect to copy number, integration site and sequence integrity. These findings provide insight into the genomic evolution of HEK293 cells and offer a foundation for further multi‐omics studies aimed at optimizing HEK293 cells for applications in biopharmaceutical production.

Stable cell lines have recently achieved recombinant adeno-associated virus (rAAV) titers comparable to the standard triple transfection approach, making them a promising alternative to plasmid-based production systems. However, whether integration of the rAAV transgene into the host genome influences packaging efficiency and vector quality remains unclear. In this study, we generated stable HEK293 cell lines carrying the rAAV transgene in their genome. rAAV production was enabled by supplying the rep/cap and helper genes on two plasmids, rendering vector genome generation dependent on the chromosomally integrated transgene. Although the stable cell lines produced a 4.5-fold lower titer of viral genomes (VGs) compared to the standard triple transfection method, VG-normalized potency was four times higher. Detailed particle characterization further revealed 3-fold lower plasmid backbone DNA packaging in rAAVs produced by stable cell lines relative to triple transfection. Consistent results were obtained from mass photometry and ELISA/ddPCR analyses for the double transfection condition, while discrepancies emerged under triple transfection. These findings emphasize the importance of functional and qualitative assessments for evaluating different rAAV production approaches.

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.