Optimized two-step electroporation process to achieve efficient non-viral-mediated gene insertion into primary T cells

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 non-viral 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 non-viral 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.