Abstract 3539: Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression

Abstract

Abstract Innovative strategies are required for cancer treatment, especially evident given that many patients relapse or do not respond to current therapeutics. Adenoviral vectors are powerful tools for gene transfer and our laboratory employs a vector where the fiber was modified to present the RGD peptide, considerably improving cell transduction. We developed a system that controls transgene expression by the PG promoter, which is responsive to p53 transcriptional activity. We utilize this system to deliver the human p53 and interferon beta cDNAs. The present study evaluated the transduction efficiency, the dependence of transgene expression on p53 status in human colorectal cancer cell lines HCT116 (p53 wt), HCT116p53-/- and HT29 (p53 mut) and the influence of the transgenes on cell viability. The vectors efficiently transduced all of the tested cell lines. Even at MOIs as low as 10, it was possible to see 42% of cells HCT116 positive for GFP using the AdRGDPGeGFP vector. Higher MOIs (50 and 100) did not present major differences in transduction efficiency (approximately 85%), but the GFP expression intensity increased approximately 14 fold. Cells were treated with doxorubicin (1μM) in order to stimulate p53 activity and when a MOI of 10 was used, 54% of cells were GFP positive with a 16 fold increase in the expression level, yet expression intensity rose 16 fold (MOI 50) and two fold (MOI 100), when compared with non-stimulated with doxorubicin. Since p53 expression is absent in the HCT116p53-/- cell line, we would not expect to observe GFP expression when using the PG promoter. However, very low level GFP activity was detected and was slightly induced upon treatment with doxorubicin, suggesting a p53-independent mechanism for transgene expression. However, restoring p53 in this cell line led to higher expression levels of GFP. We observed an important benefit mediated by our vectors (MOI of 25) for the reduction of cell viability mediated by doxorubicin, cisplatin and 5-fluorouracil (5-FU). The effect of doxorubicin was especially interesting, even at a low dose (0.625μM) the co-transduction with both the p53 and IFN-β vectors conferred a benefit higher than that seen with isolated transductions. The dose of 10μM cisplatin and 6.25μM 5-FU also provided a major benefit in reducing cell viability upon co-transduction. Both HCT116 and HCT116p53-/-responded in a similar way to gene transfer combined with chemotherapeutics, but HT29 was more sensitive to IFN-β when associated with doxorubicin (0.625μM) and cisplatin (doses range 10 - 1.25 μM). Thus, our system reveals an opportunity to improve transduction efficiency and transgene expression and, as a consequence, increase sensitivity to genotoxic stress. Moreover, the co-transfection of p53 and IFN-β may improve the efficacy of chemotherapeutics in colorectal cancer. Financial support: Sao Paulo Research Foundation (FAPESP) 2013/16074-3; 2013/25167-5. Citation Format: Paulo Roberto Del Valle, Daniela B. Zanatta, Bryan E. Strauss. Modifications of adenoviral structure and genome improves transduction efficiency and transgene expression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3539. doi:10.1158/1538-7445.AM2015-3539