307. Prostate Targeted TSTA Oncolytic Adenovirus

Abstract

Top of pageAbstract Due to the limited treatment options currently available for recurrent hormone refractory prostate cancer (HRPC), gene therapy can provide a promising tool to fight this challenging disease. We employed a prostate-targeted gene expression system to develop a safe and efficient vector-based gene delivery approach. The combination of a modified PSA promoter/enhancer and the two- step transcriptional amplification (TSTA) system enabled us to achieve robust expression of transgenes while maintaining prostate specificity. Furthermore, integration of an efficient adenovirus- mediated gene delivery approach and noninvasive imaging enabled real-time assessment of in vivo gene expression by optical CCD imaging (Zhang 2003), as well as clinically-translatable PET radionuclide imaging, applicable to both early stage androgen dependent and late stage HRPC human prostate cancer xenograft models (Sato 2005). In applying PET to monitor HSV-tk-mediated suicide gene therapy, the TSTA system exhibited effective tumor cytoxicity and remarkably lowered liver toxicity compared to constitutive CMV promoter driven system (Johnson 2005). Given the utility and flexibility of the TSTA gene expression system, we further explored the application of this method to develop a robust, prostate-restricted oncolytic adenoviral vector to treat HRPC. This oncolytic vector is designed based on our previous findings that two distinct genes can be transcribed in opposite orientations from centrally located Gal4 binding sites (ASGT 2005; Ray 2004). Hence, in our oncolytic vector, the two key viral regulatory E1A and E1B gene are driven by the Gal4VP16 activator, which in turn is controlled by an augmented prostate-specific PSA promoter. To establish the feasibility of this system, we first developed a set of bi-directional E1 vectors with centrally located Gal4 sites, ranging from 0 (negative control), 2, to 4 tandem repeats. The prostate-specific Gal4VP16 was delivered in trans by a second vector. We discovered that the level of E1 protein products, viral DNA amplification and infectious virus production in PSA- and AR-positive LNCaP cells was non-existent in the virus with 0 Gal4 repeat (G0) and proportionally increased in the G2 (2 sites) and G4 (4 sites) virus. These proof-of-concept findings led us to advance this prostate specific oncolytic technology for in vivo application. Towards this end, we generated all-in-one adenoviral constructs that carry both bi-directional E1 (G0, G2, G4) and the activator expression cassettes (inserted in E3 region) in individual viruses. Infection study showed that all-in-one TSTA oncolytic adenovirus (G2 and G4) can specifically replicate in AR positive prostate cancer cells (LAPC-4 and LNCaP) but not in non-prostate cells (e.g. HeLa and A549). Real-time PCR analysis illustrated a robust increase in the copy number of oncolytic viral genome in prostate cancer cells, and this method is applied to establish the time course and efficiency of replication in comparison to wild-type adenovirus. We foresee that this TSTA oncolytic approach to be a promising gene therapy strategy for the advanced stages of prostate cancer.