Molecular Engineering of a Two-Step Transcription Amplification (TSTA) System for Transgene Delivery in Prostate Cancer

Abstract

Gene therapy is founded on the concept that tissue-specific promoters can express heterologous genes for molecular imaging or therapeutic applications. The engineering of cell-specific enhancers to improve potency and the development of two-step transcriptional activation (TSTA) approaches have significantly improved the efficacy of transgene expression. Here we combine these technologies to create a robust, titratable, androgen-responsive system targeted to prostate cancer cells. Our "chimeric" TSTA system uses a duplicated variant of the prostate-specific antigen (PSA) gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains. We targeted the resulting activators to cells with reporter templates bearing one, two, or five GAL4 binding sites upstream of firefly luciferase. We monitored activity via firefly luciferase assays in transfected cell extracts and in live nude mice using a cooled charge-coupled device (CCD) imaging system. In this system, we found that firefly luciferase expression in prostate cancer cells can be varied over an 800-fold range. We also found that a single plasmid bearing the optimized enhancer, GAL4-VP16 derivative, and reporter expressed firefly luciferase at 20-fold higher levels than the cytomegalovirus enhancer. We discuss the implications of this strategy and its application to molecular imaging and therapy.

MicroPET imaging of Cre–loxP-mediated conditional activation of a herpes simplex virus type 1 thymidine kinase reporter gene

“…45,46 In the TSTA strategy, a potent transcriptional activator, driven by a tissue-specific promoter, activates a GAL4-VP16 fusion protein, which, in turn, drives reporter gene expression. 47,48 It is important that any reporter geneimaging approach not significantly perturb the cells/ animal models being studied. However, there is potential for the GAL4-VP16 system to be toxic to cells.…”

Molecular imaging in living subjects: seeing fundamental biological processes in a new light

“…A two-step transcriptional amplification (TSTA) method for imaging gene expression using weak promoters (i.e., most tissue-specific ones) has been described by Iyer et al (2001b) and in a follow-up study by Zhang et al (2002b). The TSTA system was used to amplify expression of Fluc and HSV1-sr39tk in a prostate cancer cell line using a duplicated variant of the prostate specific antigen gene enhancer to express GAL4 derivatives fused to one, two, or four VP16 activation domains.…”

It's not just about anatomy: In vivo bioluminescence imaging as an eyepiece into biology

“…This represents an advancement that will permit more subtle changes in transcription to be analyzed by imaging. Zhang and coworkers (92) have used such an amplification method in an androgen-responsive DNA-based therapy that is directed against prostate cancer. The strategy in this study was also to use the PSA gene enhancer in duplicate to express derivatives of the transactivator GAL4 fused to VP16 activation domains.…”