Abstract
Retroviral gene delivery is widely used in T cell therapies for
hematological cancers. However, viral vectors are expensive to
manufacture, they integrate genes in semi-random patterns, and their
transduction efficiency is highly variable. In this study, several
non-viral gene delivery vehicles, promoters, and additional variables
were compared to optimize non-viral transgene delivery and expression in
both Jurkat and primary T cells. Overall, transfecting Jurkat cells in
X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection
efficiency (63.0±10.9% EGFP+). However, the same method yielded a much
lower transfection efficiency in primary T cells (8.1±0.8% EGFP+).
Subsequent confocal microscopy revealed that a majority of the
lipoplexes did not enter the primary T cells, which might be due to
relatively low expression levels of heparan sulfate proteoglycans
(HSPGs) detected via mRNA-sequencing. PYHIN DNA sensors (e.g., AIM2,
IFI16) were also expressed at high levels in Primary T cells, which can
induce apoptosis when bound to cytoplasmic DNA. Therefore, transfection
of primary T cells appears to be limited at the level of cellular uptake
and/or DNA sensing in the cytoplasm, so both of these factors should be
considered in the development of future viral and non-viral T cell gene
delivery methods.