-(PSE-BC)-(GAL4-(VP16))-(GAL4)-sr39

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 administrations (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 optical imaging, or suicide genes such as herpes simplex virus type 1-thymidine kinase (HSV1-TK, also a reporter gene for positron emission tomography (PET)), can be used for therapeutic applications (7). HSV1-TK is a homodimer composed of two 376-residue subunits, and it functions as a key enzyme in the pyrimidine-salvage pathway to catalyze the phosphorylation of thymidine (dT) to thymidine monophosphate (dTMP) in the presence of ATP and Mg (7). Unlike mammalian thymidine kinases, HSV1-TK can efficiently phosphorylate substrates such as acycloguanosines and uracils (8). Thus, radiolabeled acycloguanosines such as 9-(4-[F]fluoro-3-hydroxymethylbutyl)guanine ([F]FHBG, [F]FPCV) or uracils such as 2’-fluoro-2’-deoxy-1-β-D-arabino-furanosyl-5-iodo-uracil ([I]FIAU) are used as HSV1-TK imaging probes for positron emission tomography (PET) or single-photon emission tomography (SPECT). As a mutant of HSV1-TK, HSV1-sr39 has five nonpolar amino acids being replaced at its active site: L159I, I160F, F161L, A162F, and L163M (9). Such remodeling in the active site leads to a substantial increase in the binding affinity to acycloguanosines; i.e., the 50% inhibition concentration of ganciclovir reduces ~300-fold. This also increases the imaging detection sensitivity; i.e., a more than two-fold enhancement has been observed in detection of [F]FHBG with HSV1-sr39 compared to that with HSV1-TK, which provides an efficient gene-reporter probe combination (HSV1-sr39-[F]FHBG) for PET imaging (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) (10). 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 (11, 12). The TSTA system is composed of two parts in general; i.e., an activator PBC-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)-sr39 (G5-sr39), which contains a reporter with five GAL4 repeats to generate a GAL4-responsive sr39 (13). 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)-sr39 (AdTSTA-sr39), a reporter gene for PET imaging of AR-responsive PSA . Such a construct can provide ~12-fold amplification (12) two steps: the PSA regulates the expression of the potent transcription activator GAL4-VP2, which in turn activates the GAL4-responsive reporter gene sr39. AdTSTA-sr39 can function effectively as a gene delivery vehicle and as a lymphotropic agent in prostate cancer and metastasis (13, 14).