Establishment of a scalable engineered cell-line platform for direct, GMP-grade production of eVLP vectors enabling streamlined generation of gene-edited CAR-T/NK cells.

Authors

Wei Lin, Jiaru Shi, Hanyi Chen, Ruikai Chai, Sha Zhu, Lili Chen, Shengshui Mo, Zhengbo Wang, Huaxiu Li, Yaru Feng, Lijun Zhao, Junhui Chen, Guohua Yu, Tao Lu, Jianxun Wang

Published in

Frontiers in immunology. Volume 17. Pages 1878099. Epub Jul 27, 2026.

Abstract

CRISPR-Cas9 has transformed the engineering of chimeric antigen receptor T (CAR-T) cells and chimeric antigen receptor NK (CAR-NK) cells; however, its clinical translation remains constrained by the high cost, batch-to-batch variability, and stringent regulatory requirements associated with current viral and electroporation-based manufacturing approaches.
We report an industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9-gRNA ribonucleoproteins (RNPs). A progenitor cell line was established by stably integrating three core modules-Gag-Pol, Gag-Cas9, and the baboon endogenous virus (BaEV) envelope-into a single HEK293T clone. Introduction of a self-inactivating (SIN) retroviral vector encoding the gRNA cassette (exemplified here by CD7) converted this progenitor into a dedicated eVLP producer within 10 days.
Using this platform, we generated CD7-knockout CAR-T/NK cells that retained robust in vitro cytotoxicity, confirming preserved functional activity. Owing to its modular architecture, the platform is readily extensible. For example, integration with Recombinant Adeno-associated Virus (rAAV) donor templates enables site-specific CAR insertion, while multiplexed eVLP cocktails allow simultaneous disruption of multiple genomic loci.
It is worth noting that this workflow eliminates the need for electroporation, reduces serum dependency, and significantly lowers the cost of reagent consumables. Collectively, this system provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.

PMID:
42577382
Bibliographic data and abstract were imported from PubMed on 11 Aug 2026.

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