Creating a built-in suicide system in Adeno associated viral constructs as a safety check mechanism for treatment of possible tumor development in long term gene therapy studies.
Adeno associated virus (AAV) is proving to be a powerful tool for gene therapy. However, a drawback of this tool reported in experimental studies is that a small percentage of the administered virus can integrate into host deoxyribonucleic acid (DNA) sequences, sometimes resulting in oncogenesis. Suicide gene therapy (SGT), a strategy for the gene-directed killing of cells, is one of the approaches that could be well utilized to augment existing gene therapies by providing a safety element in the event of tumor development. This technique has not been used in AAV based gene therapy experiments. One of the successful SGT systems utilizes Herpes Simplex virus thymidine kinase gene (HSVtk) which confers ganciclovir (GCV) sensitivity to the transduced cells. We hypothesized that placing HSVtk/GCV under the control of the HSP70 (Heat Shock Protein 70) promoter will results in apoptosis of HSVtk expressing cells, only when heat is applied. We developed a dual promoter-transgene AAV viral construct (AAV-70-tk-CAG-GFP) carrying an inducible HSP70 promoter for the expression of HSVtk and chicken β-actin (CAG) (a ubiquitous promoter), driving green fluorescent protein (GFP). Human embryonic kidney cells (HEK293 cells) were stably transfected with the study plasmid and analyzed for protein expression. The experimental and control cells were exposed to normal (37°C) and high temperature (41°C) to measure cell survival at different concentrations of GCV. The survival of stably transfected cells was markedly reduced with GCV treatment at 41°C, compared to untransfected HEK 293 cells. However, an unexpected decrease in cell survival was also noted at 37°C, indicating the promoter was also active at normal temperature. In summary, our experiment was the first step to test cell killing with SGT under the control of an inducible promoter in an AAV construct. The results showed cell killing both at high and normal temperatures. This provides a preface for future research exploring HSP70 induction physiology and use of other inducible promoters in the HSVtk/GCV suicide gene system.