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Basic enables selection-free efficient knockin of large DNA in primary human T cells
Kexin Wang1, Xiaorui Li1, Jin Li1
1Frontiers Science Center for Molecular Design Breeding (MOE), State Key Laboratory of Animal Biotech Breeding, College of Biological Sciences, China Agricultural University, Beijing 100193, China.
Summary
A new platform called BASIC efficiently inserts large DNA fragments into human T cells for gene therapy. This method achieves high knock-in efficiency without cell damage, enabling advanced cell therapies.
Area of Science:
- Biotechnology
- Molecular Biology
- Immunotherapy
Background:
- Inserting large DNA fragments into primary human T cells is challenging for gene and cell therapy development.
- Current methods often suffer from low efficiency, lack of precision, or compromise cell viability.
Purpose of the Study:
- To develop an efficient and precise method for large DNA fragment insertion into primary human T cells.
- To enable multiplex genome engineering and improve the generation of therapeutic T cells.
Main Methods:
- Utilized BASIC, a modular platform combining BaEVshort-pseudotyped virus-like particles for Cas9 RNP delivery and AAV6 donor vectors for homology-directed repair.
- Delivered Cas9 RNP and AAV6 donor vectors to primary human T cells.
Main Results:
- Achieved >85% knock-in efficiency for large DNA fragments without drug selection or electroporation.
- Preserved cell viability and enabled multiplex genome engineering.
- Edited CAR-T cells demonstrated uniform CAR expression, enhanced cytotoxicity, and complete tumor clearance in vivo.
Conclusions:
- BASIC provides a clinically scalable solution for efficient and precise genome engineering in primary human T cells.
- This platform facilitates the development of next-generation cell therapies, including CAR-T cell therapies.
- The method overcomes previous bottlenecks in T cell modification for therapeutic applications.
