Optimizing Non-Viral Gene Delivery to T Cells with Lipofectamine LTX

Retroviral gene delivery is widely used in CAR-T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In contrast, non-viral delivery methods could offer a potentially less expensive and safer alternative for CAR-T cell gene delivery. In this study, several different cationic vehicles, promoters, and additional culture conditions were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. In addition, confocal microscopy and next-generation sequencing experiments were also conducted to detect putative methods of transfection resistance in Jurkat and primary T cells. Transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency in Jurkat cells (63.0±10.9% EGFP+). However, this protocol yielded a much lower transfection efficiency (8.07±0.76% EGFP+) in primary T cells. Confocal microscopy and mRNA-sequencing experiments revealed that a majority of lipoplexes did not enter the primary T cells, perhaps due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs). Primary T cells also expressed high levels of PYHIN DNA sensors (e.g., AIM2, IFI16), which can induce apoptosis when bound to cytoplasmic DNA. Primary T cells are more resistant to transfection with Lipofectamine than Jurkat T cells. Transfection of primary T cells appears to be hindered by decreased expression of HSPGs and high expression of PYHIN DNA sensors. Both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods. Overall design: 3 cell lines, 6 samples per cell line with (3) untransfected and (3) transfected samples (n=3) (18 samples overall)