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 mutant GBMs as well as IDH1 wildtype GBMs, respectively. Due to TP53 deficiency, in gliomas tumor malignancy gradually rise and resistance to radio and chemotherapy increase. We sought to develop a selective sleeping beauty transposase system for p53-replacement based on cyclodextrin-modified poly(propylene imine) polyplexes for gene therapy of GBM.

Methods: A codon-optimized p53 coding sequence was chemically synthesized and ligated into a GFP-transposon minicircle-DNA plasmid. SB100X and p53-transposon minicircle DNA were simultaneously packaged in modular cyclodextrin-modified poly(propylene imine) hybrid polyplexes bioconjugated to a PSCA-specific single chain antibody. Specific delivery of nanoparticles was confirmed by FACS analysis of GFP in HEK293T-PSCA followed by analyzing stable transgene expression using PCR. After Zeocin-induced DNA-damage response in transposon-treated HCT116-PSCA p53-/- cells and H4-PSCA cells, appearance of phospho-Ser15-p53 and increased expression of the downstream effector molecule p21waf/cip were investigated using Western blot analysis. Long-term effects of p53-replacement were investigated in clonogenic survival assays of HCT116 p53-/- and H4-PSCA.

Results: Sleeping beauty-mediated p53 gene transfer of our modular PSCA-specific hybrid polyplex system lead to successful expression of a fully functional transgenic TP53 tumor suppressor gene as validated by Western blot analysis and a Zeocin-induced DNA-damage response. Experimental replacement of p53 in HCT116-PSCA p53-/- and H4-PSCA cells caused up to 80% decrease in clonogenic survival of HCT116-PSCA p53-/- and H4-PSCA when compared to mock-treated controls.

Conclusion: We successfully developed a targeted DNA delivery system for p53-replacement therapy. In particular, in our in vitro experiments polyplex-based transfection of SB100X and p53-minicircle transposon DNAs show significant effects for p53-gene deficient and p53 antagonists upregulated cell lines. Further work for effective non-viral delivery of the sleeping beauty and p53-transposon using nanoparticle delivery systems in primary GBM cells are mandatory for a future clinical use.