1. ¹Department of Molecular and Medical Pharmacology, University of California at Los Angeles (UCLA) School of Medicine, Los Angeles, California, USA
2. ²Department of Radiology, Stanford University School of Medicine, Stanford, California, USA
3. ³Department of Bio-engineering, Stanford University School of Medicine, Stanford, California, USA
4. ⁴Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA
5. ⁵Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, California, USA
6. ⁶BIO-X, Stanford University School of Medicine, Stanford, California, USA

Correspondence: Sanjiv S. Gambhir, Department of Radiology, 318 Campus Drive, Clark Center, E150, Stanford, California 94305-5330, USA. E-mail: sgambhir@stanford.edu

Received 9 January 2008; Accepted 22 July 2008; Published online 2 September 2008.

Abstract

Promoters that limit transgene expression to tumors play a vital role in cancer gene therapy. Although tumor specific, the human Survivin promoter (pSurv) elicits low levels of transcription. A bidirectional two-step transcriptional amplification (TSTA) system was designed to enhance expression of the therapeutic gene (TG) tumor necrosis factor-–related apoptosis-inducing ligand (TRAIL or TR) and the reporter gene firefly luciferase (FL) from pSurv. An adenoviral vector carrying the enhanced targeting apparatus (Ad-pSurv-TR-G8-FL) was tested for efficiency and specificity of gene expression in cells and in living animals. Compared to the one-step systems (Ad-pSurv-FL or Ad-pSurv-TR), the bidirectional TSTA system showed tenfold higher expression of both the therapeutic and the reporter gene and their expression correlated in cells (R² = 0.99) and in animals (R² = 0.67). Noninvasive quantitative monitoring of magnitude and time variation of TRAIL gene expression was feasible by bioluminescence imaging of the transcriptionally linked FL gene in xenograft tumors following intratumoral adenoviral injection. Moreover, the TSTA adenovirus maintained promoter specificity in nontarget tissues following tail vein administration. These studies demonstrate the potential of the bidirectional TSTA system to achieve high levels of gene expression from a weak promoter, while preserving specificity and the ability to image expression of the TG noninvasively.