Plasmid DNA cationic non-viral vector complexes induce cytotoxicity-associated PD-L1 expression up-regulation in cancer cells in vitro

Non-viral vectors are promising nucleic acid carriers which have been utilized in gene-based cancer immuno-therapy. The aim of this study is to compare the transfection efficiency and cytotoxicity of three cationic non-viral vectors namely Polyethylenimine (PEI), Lipofectamine 2000 (LPF) and stable nucleic acid lipid particles (SNALPs) of different lipid compositions, for the delivery of plasmid DNA (pDNA) expressing immunostimulatory molecules, OX40L or 4-1BBL, to cancer cells in vitro. The results indicate that PEI and LPF are efficient vectors for pDNA delivery with high transfection efficiency obtained. However, pDNA-PEI and pDNA-LPF complexes up-regulated the expression of programmed death ligand-1 (PD-L1) and induced significant cytotoxicity in both B16F10 and CT26 cell lines. The up-regulation of PD-L1 expression induced by pDNA-PEI and pDNA-LPF complexes was independent of cancer cell line, nor was it linked to the presence of GpC motifs in the pDNA. In contrast, the use of biocompatible SNALPs (MC3 and KC2 types) resulted in lower pDNA transfection effi-ciency, however no significant up-regulation of PD-L1 or cytotoxicity was observed. A strong correlation was found between up-regulation of PD-L1 expression and cytotoxicity. Up-regulation of PD-L1 expression could be mitigated with RNAi, maintaining expression at basal levels. Due to the improved biocompatibility and the absence of PD-L1 up-regulation, SNALPs represent a viable non-viral nucleic acid vector for delivery of pDNA encoding immunostimulatory molecules. The results of this study suggest that PD-L1 expression should be monitored when selecting commercial transfection reagents as pDNA vectors for cancer immunotherapy in vitro.