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<p>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 -&#xa0;commonly used in CHO cells&#xa0;- 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 (&gt;1x10<sup>11</sup> 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.</p>

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Streamlined rAAV HeLaS3 producer cell line generation via GS selection

  • 期刊论文

  • 出版模式：

    开放获取

  • 发表日期：

    2026年1月9日

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  • Mariana Antunes,
  • Filipa Moura,
  • Ivy Rose Sebastian,
  • Paula Alves,
  • Patrícia Gomes-Alves,
  • Jose M. Escandell

摘要

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.