-(PSE-BC)-(GAL4-(VP16))-(GAL4)-Fluc

Adenoviruses (Ads) comprise a non-enveloped icosahedral protein shell (capsid) 70–100 nm in diameter surrounding an inner vial genome core (1). Human Ads consist of 51 distinct serotypes that are classified into six subgroups (species) designated A–F (2). Some serotypes such as serotypes 2 (Ad2) and 5 (Ad5) of species C only induce a mild, non-oncogenic respiratory infection, making them suitable for development of Ad-based vaccines and gene delivery vehicles for systematic administration (1). Their core genome, a linear, double-stranded DNA of ~36 kb, contains five early transcription units (E1A, E1B, E2, E3, and E4), four intermediate units (IVA2, IX, VAI, and VAII), and one later transcription unit (2). The functions in the early transcription regions have been well identified,: E1A activates cell cycles and initiates DNA replication; E1B blocks apoptosis induced by E1A activity; E2 facilitates viral DNA replication; E3 modulates host immune responses; and E4 regulates DNA replication, mRNA transport, and apoptosis (3). As a gene delivery vehicle, the genome core in Ad2 or Ad5 is usually modified with recombinant transcription activators (RTAs), in which foreign DNA with a size of up to 7.5 kb can be inserted in the deleted E1 or E3 regions in the Ads (4). Such genetic modification can incorporate ligands that can recognize specific cellular receptors and/or block the adenoviral naïve receptor (coxsackie-adenovirus receptor (CAR)), so that their tissue specificity is enhanced significantly. For example, the gene for the androgen receptor (AR) regulates the growth of prostate epithelial cells, induces the expression of prostate-specific antigen (PSA), and controls the growth of prostate cancer in the early phase. The AR gene can be introduced to the adenoviral genome to target AR-responsive prostate cancer or metastasis (5). Furthermore, reporter genes such as firefly luciferase (Fluc) (6) can be included for imaging, or suicide genes such as herpes simplex virus type 1-thymidine kinase (HSV1-tk) can be used for therapeutic applications (7). As an oxygenase (Mw = 62 kDa), Fluc oxidizes the heterocyclic substrate d-luciferin to oxyluciferin injected into the body and emits light in the wavelength range of 400–620 nm (8). The transcriptional activity of adenoviral genes can be amplified several orders of magnitude with the use of the GAL4-VP2 fusion protein, where GAL4 is a DNA-binding domain (147 amino acids) and VP2 is a two-tandem repeat of the herpes simplex virus VP16 acidic activation domain (78 amino acids) (9). The two-step transcriptional amplification (TSTA) system is a novel recombinant transcription activation approach for designing a PSA promoter/enhancer, which can provide as high as ~800-fold enhancement in transcription activity compared with its conventional analog (10, 11). The TSTA system is composed of two parts in general; i.e., an activator BC-VP2 that contains a PSA promoter with a duplicated ARE4 enhancer core (each core has four binding sites for AR) to control the expression of the chimeric protein GAL4-VP2 and provide cell-specific binding, and the reporter (GAL4)-Fluc (G5-FL), which contains a reporter with five GAL4 repeats to generate a GAL4-responsive Fluc signal (6). The two parts are linked in a head-to-head orientation and are inserted into the E1 region of Ad5 to produce -(PSE-BC)-(GAL4-(VP16))-(GAL4)-Fluc (AdTSTA-FL), an optical agent for imaging AR-responsive PSA. Such a construct can provide ~50-fold amplification (11) two steps: the PSA regulates the expression of the potent transcription activator GAL4-VP2, which in turn activates a GAL4-responsive reporter Fluc. AdTSTA-FL can function effectively as a gene delivery vehicle and as a lymphotropic agent in prostate cancer and metastasis (6, 12).