gms | German Medical Science

Objective: Hot spot mutations and deletions of the tumor suppressor p53 are detected in 35% of IDH1 wildtype and in more than 65% of IDH1-mutant glioblastoma (GBM). Accelerated degradation of TP53, leading to loss of function is also considered to be caused by deletion or mutation of PTEN and LOH on chromosome 10q in IDH1, respectively. Due to TP53 deficiency, tumor malignancy gradually increases and resistance to radio and chemotherapy increases. We sought to develop an experimental p53-replacement therapy using a retroviral system and a sleeping beauty transposase system, respectively, for gene therapy of GBMs.

Methods: A codon-optimized p53 coding sequence was chemically synthesized and ligated into pHATtrick-puro lentiviral vector or ligated into a transposon minicircle-DNA plasmid. Viral transduction or (poly)ethylenimine-mediated simultaneous transfection of SB100X sleeping beauty and transposon minicircle DNA were utilized for p53 replacement. HCT116 p53-/- and HT1080 cells with knockdown of p53 were employed to analyze stable p53 transgene expression following genetic transfer of p53 using PCR and were used to verify a functional p53 response upon DNA damage. Transgenic expression and TP53-mediated damage response was confirmed using indirect immunofluorescence analysis of phospho-gammaH2AX in Zeozin-treated cells and was further investigated using Western blot analyses for TP53 and p21waf/cip. Long term effects of p53-replacment were investigated in clonogenic survival assays of HCT116 p53-/-, HT1080 cells and primary cell cultures from GBM samples.

Results: Retroviral and sleeping beauty-mediated p53 gene transfer lead to expression of a fully functional transgenic TP53 tumor suppressor gene as validated by PCR analysis and a Zeozin-induced DNA-damage response. Experimental replacement of p53 in HCT116 p53-/-, HT1080 cells and primary cell cultures from GBM samples caused up to 90% decrease in clonogenic survival of HCT116 p53-/-, HT1080 cells and primary cell cultures from GBM samples when compared to mock-treated controls.

Conclusion: We successfully developed a p53-replacement for treatment of glioblastoma. In particular, in our in vitro experiments retroviral transduction was superior when compared to transfection of SB100X and p53-minicircle transposon DNAs. Further work for effective non-viral delivery of the sleeping beauty and p53-transposon, in particular in using nanoparticle delivery systems are mandatory for a future clinical use.