In vivo bioluminescent monitoring for therapeutic intervention by HSV1-tk/GCV system using hTERT RNA targeting trans-splicing ribozyme

Objectives HSV1-tk/GCV system using adenovirus mediated hTERT RNA targeting trans-splicing ribozyme suggested as a useful strategy for tumor targeted gene therapy. This study aims to visualize the therapeutic effect of this treatment system in vitro and in vivo.

Methods Adenoviral vector(Ad-CRT) containing the hTERT targeting trans-splicing ribozyme with the downstream HSV1-tk gene driven by the CMV promoter was infected to firefly luciferase expressing human colorectal adenocarcinoma cells(LS174T-Luc). To determine the TK expression in Ad-CRT infected cell, cellular uptake with [¹²⁵I]IVDU was measured according to time intervals. After infection of Ad-CRT at 150 MOI for 24 h, cells were treated with various concentration of GCV for 96 h. The cytotoxic effects were determined using MTT assay and luciferase activity assay. 2.5×10⁸pfu of Ad-CRT were intratumorally injected in tumor-bearing mice. GCV treatment was performed through intraperitoneal injections (50mg/kg, daily) for 10 days. Therapeutic effect was evaluated using tumor size measurement and bioluminescent imaging.

Results Significant uptake of [¹²⁵I]IVDU was observed in Ad-CRT infected LS174T-Luc cells. Luciferase activity assay showed the cytotoxic effect of GCV in Ad-CRT infected LS174T-Luc as dose dependent manner in vitro. Inhibition of tumor growth was observed in tumor-bearing mice treated with Ad-CRT/GCV by tumor size measurement.

Conclusions In conclusion, we could monitor the therapeutic effect of Ad-CRT/GCV system using bioluminescent imaging and tumor size measurement.