Targeting validation of a heat-induced, tumor-specific gene therapy vector

Objective: To verify tumor- and heat-specificity to hyperthermia stimulation of a gene therapy vector containing the gene PNP regulated by the eight heat shock elements (8HSEs) and the hTERT promoter. Methods: (1) The expression of hTERT was measured by RT-PCR, Western blot and immunocytochemistry assays. (2)The recombinant lentiviral vector pLVX-8HSEs-hTERTp-PNP-3FLAG(8HhP) containing an hTERT promoter and 8 copies of the HSEs, which was constructed in our previous study, was collected. 8HhP was transfected into hTERT⁺ SW480 cells, hTERT^- MKN28 cells and hTERT^- MRC-5 cells; then the tumor- and heat-specificity was verified by immunofluorescence. Western blot and RT-PCR were used to examine the expression of PNP under heated conditions. Results: hTERT mRNA and protein expressions were high in SW480 cells, but low or negative in MKN28 and MRC-5 cells. The expressions of PNP protein and mRNA in 8HhP/SW480 cells were obviously increased only under heated conditions, which was confirmed by Western blot, RT-PCR and immunofluorescence. In contrast, the expressions of PNP protein and mRNA in 8HhP/MKN28 and 8HhP/MRC-5 cells were low or negative even under heated conditions. Conclusion: The hTERT promoter modified by 8HSEs can increase the gene therapy vector specificity to hyperthermia and tumors.