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Background <p>Recombinant adeno-associated viruses (rAAVs) are the vectors of choice for gene therapy applications due to their favorable safety and efficacy profiles. However, current production platforms—particularly transient transfection-based HEK293 systems—have scalability challenges, primarily due to the high cost of GMP-grade plasmid DNA and low specific productivity when scale up is required. This has become a significant bottleneck in the commercialization of rAAV-based gene therapy products, prompting recent market withdrawals or limited commercialization of gene therapy products based on rAAV vectors. In this context, stable producer cell lines (PCLs), such as HeLaS3-based systems, offer a promising alternative for cost-effective and scalable rAAV manufacturing. Nonetheless, these systems are still in early stages of development and often yield lower titers.</p> Results <p>To address these limitations, we employed a CRISPR activation (CRISPRa) screen to identify genetic regulators that enhance rAAV production. This approach revealed several pathways related to protein trafficking and immune response as key contributors to rAAV biogenesis. Notably, we identified <i>CEBPA</i> gene as a master regulator in this context. Overexpression of <i>CEBPA</i> reprogrammed the host transcriptome, activating immune-related pathways and enhancing cellular metabolism. Importantly, when combined with bioprocess intensification strategies, i.e. perfusion, <i>CEBPA</i>-overexpressing cells were able to maintain cell-specific production yield at higher cell density while maintaining high vector quality. This combined approach led to up to 10-fold increase in volumetric productivity compared to non-modified parental cells.</p> Conclusions <p>These findings will contribute to the development of a robust and scalable production platform that enhances vector yield without compromising quality. Our approach addresses a critical barrier in gene therapy manufacturing offering a practical path towards broader clinical and commercial viability.</p>

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Enhancing gene therapy vectors manufacturing: CEBPA as a master regulator of rAAV biogenesis

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年1月20日

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  • Filipa Moura,
  • Mariana Antunes,
  • Ricardo Correia,
  • António Roldão,
  • Patrícia Gomes-Alves,
  • Paula Marques Alves,
  • Jose M. Escandell

摘要

Background

Recombinant adeno-associated viruses (rAAVs) are the vectors of choice for gene therapy applications due to their favorable safety and efficacy profiles. However, current production platforms—particularly transient transfection-based HEK293 systems—have scalability challenges, primarily due to the high cost of GMP-grade plasmid DNA and low specific productivity when scale up is required. This has become a significant bottleneck in the commercialization of rAAV-based gene therapy products, prompting recent market withdrawals or limited commercialization of gene therapy products based on rAAV vectors. In this context, stable producer cell lines (PCLs), such as HeLaS3-based systems, offer a promising alternative for cost-effective and scalable rAAV manufacturing. Nonetheless, these systems are still in early stages of development and often yield lower titers.

Results

To address these limitations, we employed a CRISPR activation (CRISPRa) screen to identify genetic regulators that enhance rAAV production. This approach revealed several pathways related to protein trafficking and immune response as key contributors to rAAV biogenesis. Notably, we identified CEBPA gene as a master regulator in this context. Overexpression of CEBPA reprogrammed the host transcriptome, activating immune-related pathways and enhancing cellular metabolism. Importantly, when combined with bioprocess intensification strategies, i.e. perfusion, CEBPA-overexpressing cells were able to maintain cell-specific production yield at higher cell density while maintaining high vector quality. This combined approach led to up to 10-fold increase in volumetric productivity compared to non-modified parental cells.

Conclusions

These findings will contribute to the development of a robust and scalable production platform that enhances vector yield without compromising quality. Our approach addresses a critical barrier in gene therapy manufacturing offering a practical path towards broader clinical and commercial viability.