Optimized two‐step electroporation process to achieve efficient nonviral‐mediated gene insertion into primary T cells

FEBS Open Bio

Diane Tkach¹  Ming Yang¹  Alex Boyne¹  Selena Kazancioglu¹  Alexandre Juillerat¹  Laurent Poirot²  Philippe Duchateau²  Aymeric Duclert² 

[1] Cellectis Inc New York NY USA;Cellectis Paris France;

关键词: chimeric antigen receptors;    double‐stranded break;    gene editing;    nonviral vectorization;    TALEN;   

DOI  :  10.1002/2211-5463.13292

来源: DOAJ

【 摘 要 】

The development of gene editing technologies over the past years has allowed the precise and efficient insertion of transgenes into the genome of various cell types. Knock‐in approaches using homology‐directed repair and designer nucleases often rely on viral vectors, which can considerably impact the manufacturing cost and timeline of gene‐edited therapeutic products. An attractive alternative would be to use naked DNA as a repair template. However, such a strategy faces challenges such as cytotoxicity from double‐stranded DNA (dsDNA) to primary cells. Here, we sought to study the kinetics of transcription activator‐like effector nuclease (TALEN)‐mediated gene editing in primary T cells to improve nonviral gene knock‐in. Harnessing this knowledge, we developed a rapid and efficient gene insertion strategy based on either short single‐stranded oligonucleotides or large (2 Kb) linear naked dsDNA sequences. We demonstrated that a time‐controlled two‐step transfection protocol can substantially improve the efficiency of nonviral transgene integration in primary T cells. Using this approach, we achieved modification of up to ˜ 30% of T cells when inserting a chimeric antigen receptor (CAR) at the T‐cell receptor alpha constant region (TRAC) locus to generate ‘off‐the shelf’ CAR‐T cells.

【 授权许可】

Unknown