638. The Optimal Placement and Orientation of the Bipartite Two-Step Transcription Amplification (TSTA) Expression System in Adenoviral Vectors for Prostate Cancer Gene Therapy (2006)

A significant challenge for successful gene therapy is to achieve a high-level of specific gene expression. However, tissue-specific promoters are much weaker than strong viral promoters (CMV). We developed a two-step transcriptional activation (TSTA) system to amplify the prostate-specific PSA promoter to levels comparable or higher than CMV while maintaining prostate specificity. The TSTA system consists of two parts, one containing a modified prostate-specific promoter (PSE-BC) driving the expression of a chimeric activator (GAL4-VP16), and a target construct containing five GAL4-binding sites upstream of the gene of interest. Recombinant adenoviral vectors (Ad) harboring the bipartite prostate-specific TSTA expression cassettes in a head-to-head orientation (H-H TSTA) achieve activity exceeding that of CMV, and cell specificity and androgen responsiveness in pre-clinical models, as measured by noninvasive bioluminescence and microPET imaging. Our previous data also indicated that the configuration of the two TSTA components could impact its regulation. Hence, we modified the configuration of the TSTA Ad vectors to further improve their specificity and activity. The new TSTA configurations of activator and reporter cassettes inserted into the same Ad include (1) an opposite tail-to-tail orientation (T-T); (2) separation by ∼20kb (activator in ΔE3 and reporter in ΔE1; E1/E3), and (3) separation by a 600-bp insulator sequence (INS). Preliminary characterization comparing to the original H-H TSTA suggests that each configuration exhibits different effects on basal gene expression, activity, and specificity. The maximal androgen-induced activities of T-T, H-H, and INS TSTA vectors are comparable (within a 2-fold difference) and they are slightly higher than E1/E3 ( 2 to 3-fold lower). The most dramatic difference observed is that the E1/E3 vector exhibits extremely low basal activity in absence of androgen compared to the other 3 constructs. Hence, we observed an androgen induction for E1/E3 vector in the 1000-fold range as compared to 10 to 100-fold range for all other constructs. Preliminary data also indicated that this low basal activity of E1/E3 also enhances cell-specificity (i.e. lower activity in non-prostate cells) and detailed characterization is in progress. Our findings support that the orientation and placement of the two components of TSTA in the Ad genome impact the mechanism of transcriptional activation. Further developments of TSTA vectors include bi-directional configurations to direct more effective multi-gene therapy or coupled imaging/therapeutic strategies in a single Ad. Therapies under development include using the E1/E3 for directing expression of a therapeutic gene (p27WT or p27T187A, a mutant more resistant to proteosome degradation) and a firefly luciferase reporter gene in a bidirectional manner. We believe that coupling prostate-targeted gene therapy with non-invasive imaging will afford us a sensitive, real-time assessment of therapeutic activity in the living subject.