Glioblastoma (GBM) represent the most common primary adult brain tumors. Despite multimodal treatment, most GBMs ultimately recur. A promising strategy for GBM treatment is suicide gene therapy, employing genes encoding cytotoxic proteins. The truncated form of Pseudomonas exotoxin A, known as PE38, encompasses an enzymatically active C-terminal domain that facilitates ADP-ribosylation and subsequent inactivation of elongation factor 2 (EF2). This molecular cascade inhibits protein synthesis and ultimately causes cell death. In this study, we exploited the ability of PE38 as a suicide gene for the treatment of GBM.

To achieve sustained PE38 transgene expression, we flanked its expression cassette with Sleeping Beauty (SB) inverted terminal repeats (ITRs). Simultaneous DNA was introduced mediated by polyethyleneimine (PEI) encoding a hyperactive form of the SB transposase (SB100X). SB100X recognized the ITRs and enabled genomic integration via a cut-and-paste mechanism. To address safety concerns associated with DNA delivery, such as non-specific cytotoxic effects on cells both the transposon and SB100X expression cassettes were flanked by attB and attP recognition sites. This design enabled the use of the bacterial PhiC31 integrase to generate minicircle DNA devoid of bacterial backbones. PE38 protein expression was examined in 293FT-diphtamide 1 knock out (DHP1KO) cells by Western blot analysis. The anti-tumor efficacy of exotoxin PE38 suicide gene therapy was investigated in both permanent (U87-MG, H4) and primary (HT18584, HT16360-1) GBM cell lines, employing comprehensive analyses of cell viability and clonogeneic survival.

Exotoxin PE38 was successfully expressed in 293FT-DHP1KO cells, as confirmed by Western blot analysis, where the absence of diphthamide, a conserved modification on eEF2, prevented ADP-ribosylation of EF2. Employing exotoxin PE38 suicide gene therapy on GBM cell lines resulted in a substantial reduction in cell viability, reaching up to 80% for the H4 cell line 7 days post-treatment. Moreover, clonogenic survival exhibited a remarkable decrease of up to 99% in both H4 and HT18584 cell lines 14 days after treatment.

Our findings demonstrate the feasibility of an exotoxin PE38 suicide gene therapy for treating human GBM cells. Further research is essential to address gene delivery and to investigate anti-glioma effects of this therapy in pre-clinical in vivo studies.