344. Recombinant Adeno-Associated Virus 2 Suicide Vectors for the Treatment of Human Sarcomas and Mesotheliomas

Abstract

Top of pageAbstract Although great efforts have been made to improve conventional therapy for sarcoma and malignant mesothelioma, the median survival time of these entities after appearance of clinical symptoms stays poor. Effective locoregional therapy using viral vectors that contain a suicide gene may be an alternative treatment strategy. For sarcoma, we previously reported the highest susceptibility for recombinant adeno-associated virus 2 (rAAV-2) vectors in human connective tissue sarcoma cells (HS-1). Now we confirm our findings in five further human sarcoma cell lines: fibrosarcoma (HT-1080), Ewing's sarcoma (RD-ES), Askin's tumor (SK-N-MC), rhabdomyosarcoma (A-204) and soft tissue sarcoma (WSKL-1). Furthermore, we found that rAAV-2 also achieved both high transduction rates and GFP expression levels in human mesothelioma (H-Messo-1, MSTO-211H and NCI-H-28). Among rAAV-2-constructs containing different promotors, after transduction, the vector with the elongation factor 1-alpha (EF1a) promotor showed the highest expression rates in both the sarcoma and mesothelioma cell lines. To ensure the use of constant vector particles number, all stocks were titrated using both the functional and our real-time PCR-based titration assay. Several new thymidine kinase (TK) gene-containing vectors under control of either the Cytomegalovirus or the elongation-factor 1 alpha (EF1a) promoter were cloned and tested. A higher expression level of the transgene was observed in the sarcoma and mesothelioma lines when using the EF1a-suicide gene-containing vectors. For the sarcomas, we were able to show a complete eradication of all rAAV-EF1a-TK/eGFP (contains a thymidine kinase/enhanced green fluorescent protein fusion protein) transduced tumor cells following exposure to ganciclovir (2.5 μg/ml) in vitro, while at this dose level >90% of mock-transduced tumor cells survived. Using mesotheliomas, a nearly complete eradication (>2 log) of transduced and GCV-treated cells could be obtained using this vector. Xenotransplantation tumor models (i.p., s.c., i.v.) for both the human sarcoma and mesothlioma cell lines were established in nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice. In proof-of-principle experiments, mice transplanted with rAAV-TK/eGFP-transduced and ganciclovir-exposed sarcoma or mesothelioma tumor cells survived >5 months while in the non-transduced group all mice had died 1 month after inoculation. Currently, further animal experiments using this suicide system are ongoing. The data shown here hold promise for further development of AAV-2-based suicide gene therapy of both soft tissue sarcoma and mesothelioma towards a future clinical application.