Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a target for cancer therapy because of its ability to induce apoptosis in malignant cells while not in most normal cells and tissues. However, its potential toxicity on some normal human cells (e.g. hepatocytes) has been questioned, but the mechanism of the related toxicity still hangs in doubt. Furthermore, the physiological function of TRAIL is much less well understood. From a clinical point of view, one of the key issues in successfully implementing cancer gene therapies or biological therapies in the clinical setting is to be able to regulate gene expression strictly and consistently as and when it is needed.
In the present study, we introduced the Tet-On regulatable gene expression system into rAAV vector (AAV-TRE-TRAIL&AAV-Tet-On) to control the soluble TRAIL expression and evaluate the efficacy in cancer gene therapy. We observed that the expression and secretion of sTRAIL could be strictly controlled by the Tet-On system in both normal and cancer cells. Transduction of various cancer cell lines with AAV-TRE-TRAIL&AAV-Tet-On under the presence of Dox resulted in significant cell death by apoptosis in SMMC-7721, SMMC-7402 liver cancer cells, and A549 lung cancer cells, but not in HepG2 liver cancer cells (Figure ). The resistance of HepG2 to AAV-TRE-TRAIL&AAV-Tet-On might be a result of inefficient of rAAV transduction or intracellular distinguishable events with TRAIL and its receptors in the cells.
Intravenous injection of rAAV in the cancer mouse models showed that sTRAIL protein was predominantly expressed in liver and tumor tissue under induction condition (Figure ). The tumor weights and volumes were obviously smaller in the animals fed with Dox compared with those without Dox. As a result, the expression could be shut down when side effects appear so as to avoid serious adverse events.
To confirm whether there is an excellent target gene expression mediated by the AAV vector with the Tet-On gene expression system, we established a positive control of rAAV vectors with a CAG promoter, which has shown a good performance in AAV-mediated gene therapy [17
]. Data in our research showed the intensity of rAAV-TRE&rAAV-Tet-On mediated target gene expression under the induction condition was similar to that of rAAV-CAG, indicating that the Tet-On gene expression system has considerable capacity in controlling target gene expression.
The Tet-On system offers many advantages over other regulable gene expression systems [30
]. The inducer Dox, an analogue of tetracycline, has been used as an antibiotic for decades and it has been well characterized in the clinical setting. It is nontoxic at doses required for gene activation in preclinical and clinical studies, and the margin of safety is high. Dox is metabolized and cleared from the body rapidly, making it an ideal drug for the rapid increase in expression, long-term expression, and rapid decrease in expression of the desired transgene. The components of the Tet-On system recognizes unique fragment of DNA, and Dox does not interfere with native proteins, reducing the potential of serious side effects.
However, a limitation of the approach is that two AAV vectors were used as a mixture. One contained TRAIL regulated by TRE, and the second contained rtTA activated by Dox. If the two AAV do not co-localize equally in tumors, then TRAIL mediated killing would not be optimized. Many researchers have attempted to construct TRE and rtTA into one vector to improve the efficiency of gene transduction. We also tried this approach to ligate rtTA, TRE promoter and TRAIL gene in the rAAV vector, therefore, forms a single rAAV vector, expecting to improve the expression intensity of the transgene. However, this approach challenges the limits of rAAV packaging capacity and causes difficulties in virus packaging. In addition, we found that the cells transduced with the single vector express the transgene in the absence of Dox (data not shown), known as gene expression leakage in regulable gene expression system. Instead, a dual rAAV vector system in which the TRE-TRAIL and rtTA were constructed into two rAAV vectors respectively provided a similar intensity in transgene expression and much more strict controllability compared with the single vector system.