Abstract
The high cost and complexity of manufacturing recombinant adeno-associated virus vectors continue to limit the broader application of gene therapies, which offer life-changing potential for individuals affected by genetic diseases. Although stable producer cell lines represent a scalable and cost-effective alternative to transient transfection methods, their development is often delayed by inefficient selection strategies and extended timelines. In this study, we present a novel application of the glutamine synthetase-based selection system - commonly used in CHO cells - to a HeLaS3-based rAAV production platform. By generating glutamine synthetase-knockout HeLaS3 cells via CRISPR-Cas9 and applying glutamine deprivation under serum-free conditions, we significantly streamlined the PCL generation process, reducing the timeline to approximately two months while maintaining rAAV productivity (>1x1011 vg/mL) and product quality (~70% full capsids). This work establishes a robust and scalable workflow for rAAV manufacturing, with the potential to enhance accessibility and reduce viral vector production costs for applications in gene therapy.
Data availability
All research data and methods presented in the main and supplementary figures are available from the lead contact upon reasonable request. Correspondence and requests for materials should be directed to J.M.E. (jose.escandell@ibet.pt).
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Acknowledgements
The author(s) gratefully acknowledge the members of the laboratory for their insightful discussions and constructive feedback throughout the development of this work.
Funding
This work was supported by the Research Unit UID/04462: iNOVA4Health – Programme in Translational Medicine, financially supported by Fundação para a Ciência e Tecnologia / Ministério da Educação, Ciência e Inovação (PTDC_BTM_ORG_1383_2020), the Associate Laboratory LS4FUTURE (LA/P/0087/2020) and the AAVscreen iBETXplore Grant. J.M.E is funded by Stimulus of Scientific Employment, Individual Support program (2020. 01216.CEECIND) and F.M. by PhD fellowship 2022.11494.BD from FCT. I.R.S. appreciate support by the FWF PhD Program Grant https://doi.org/10.55776/W1224 “Biotechnology of Proteins – BioTop”.
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M.A writing – original draft, investigation, methodology, visualization, and formal analysis. F.M., investigation, methodology, visualization, and formal analysis. I.R.S investigation, methodology, and formal analysis. P.M.A, supervision and funding acquisition P.G.A. supervision, project supervision, project administration, funding acquisition. J.M.E., writing – original draft, project conceptualization, supervision, project supervision, investigation, funding acquisition. All authors: writing – review & editing.
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Antunes, M., Moura, F., Sebastian, I.R. et al. Streamlined rAAV HeLaS3 producer cell line generation via GS selection. Sci Rep (2026). https://doi.org/10.1038/s41598-025-34826-2
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DOI: https://doi.org/10.1038/s41598-025-34826-2
