154. Tetracycline-Inducible HSV Amplicon Vectors with Expanded Range of Gene Expression Regulation

• Published in: Molecular therapy Vol. 13; no. S1; p. S60
• Main Authors: Comer, Laryssa A., Fernandez, Juliet L., Camp, Sara M., Tebbets, Jessica C., Breakefield, Xandra O., Sena-Esteves, Miguel
• Format: Journal Article
• Language: English
• Published: Milwaukee Elsevier Limited 01.05.2006
• Subjects: Apoptosis; Artificial chromosomes; Cell growth; Fibroblasts; Gene expression; Herpes viruses; United Kingdom > UK
• ISSN: 1525-0016; 1525-0024
• DOI: 10.1016/j.ymthe.2006.08.177

In this study, we developed several novel tetracycline(tet)-regulated Herpes Simplex Virus-1 (HSV) amplicon vectors by introducing various combinations of tet-dependent transcriptional silencers and activators. Also, to reduce undesirable baseline gene expression, most vectors incorporated insulator elements flanking the gene expression cassette, which carried the Firefly luciferase gene (FLuc) driven by a silencer/activator-responsive promoter. To determine which constructs were most effective at regulating gene expression, we tested the luciferase activity of transduced human glioblastoma Gli-36 cells in vitro, in the presence or absence of doxycycline (Dox). Induction levels for the twenty-six vectors tested ranged from 154-fold to 6000-fold, with the best vector (HET14B-FLuc) displaying over 5-fold higher luciferase activity than a control HSV amplicon vector constituitively expressing luciferase. Dose response analysis showed that gene expression could be regulated over a wide range of Dox concentrations, and that 1000-fold induction could be achieved at 10 ng/mL Dox. To further demonstrate the regulation capabilities of this system, we replaced the luciferase gene in HET14B-FLuc with the gene for reversed caspase-3, an auto-catalytic version of wild-type caspase-3, an executioner of apoptosis. WST cell proliferation assays demonstrate that Gli-36 cells transduced with this vector (MOI 0.5-2) undergo rapid apoptosis upon Dox induction, with almost no viable cells remaining 6 hours post-induction. Non-induced cells infected with the same vector, as well as induced and non-induced cells infected with HET14BFLuc control vector, continued growing throughout the 36 hour assay. Application of the pan-caspase inhibitor Z-VAD-FMK at the time of induction prevented apoptosis in cells infected with the caspase-3 vector, and cell proliferation levels were similar to those in control groups. Western blot analysis, probing for poly-(ADP-ribose)-polymerase, which is cleaved during apoptosis, confirm that cells infected with caspase-3 vector undergo massive apoptosis upon Dox induction, while control groups exhibit a low baseline level of cell death in all samples. Currently we are evaluating the therapeutic potential of the caspase-3 vector in vivo by direct injection into subcutaneous Gli-36 tumors constitutively expressing Fluc. Subsequent tumor growth in the presence or absence of Dox will be assessed by bioluminescence imaging of tumor- associated FLuc.