IIBBA   05544
INSTITUTO DE INVESTIGACIONES BIOQUIMICAS DE BUENOS AIRES
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
. Improving the lytic capacity of a conditionally replicative adenoviruses by incorporating promoter elements responsive to tumor environmental conditons
Autor/es:
DIEGO L. VIALE; MARIA VERONICA LOPEZ; EDUARDO A. CAFFERATA; DAVID GOULD; YUTI CHERNAJOVSKY; OSVALDO L. PODHAJCER
Reunión:
Congreso; 13th Annual Meeting of the American Society of Gene & Cell Therapy; 2010
Institución organizadora:
American Society of Gene & Cell Therapy
Resumen:
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Conditionally Replicative Adenoviruses (CRAds) are a new modality for cancer therapy. We have previously reported that a new CRAd (Ad-F512) where E1A transcription is driven by a 0.5 Kb fragment of the SPARC promoter (SPPr) exhibited strong killing effect over established human melanomas xenografted in nude mice. However, its oncolytic effect was strongly reduced on established tumors made of a mix of melanoma cells and human fibroblasts (Lopez MV et al, PLOS One, 2009). In order to improve the therapeutic efficacy of Ad-F512 we hypothesized that addition of DNA sequences containing responsive elements to different patho-physiological conditions that characterize tumor tissue, such as hypoxia and inflammation, would potentiate its replication and, therefore its oncolytic activity. We constructed twelve different chimeric promoters containing SPPr combined with a Hypoxia-Response Element (HRE), an NF?B-response element (NF?B) or both and compared its transcriptional activity in luciferase expressing plasmids. Based on their activity/specificity ratio in different malignant and stromal cells we selected the chimeric promoter HRE-SPPr where HREs were placed upstream of SPPr and a triple chimeric promoter NFkB-SP(HRE)Pr where the NFkB responsive elements were placed upstream of SPPr and HRE was placed inside SPPr. We further evaluated the transcriptional activity of the chimeric promoters in an adenovirus backbone using luciferase as a reporter gene and observed 2-10 fold induction of luciferase activity under hypoxia of both chimeric promoters and a 5-fold induction under TNF? treatment of NFkB-SP(HRE)Pr in tumor cells. Based on these results, we constructed two CRAds (Ad-HRE-SPPr and Ad-NFkB-SP(HRE)Pr) where E1A transcription was driven by the chimeric promoters and evaluated the cytopathic effect (CPE), viability and replication rate. Compared with Ad-F512 we observed a slight increase of oncolytic effect under normoxic conditions over melanoma cells. However, the best replication rate was observed under hypoxic conditions where both CRAds exhibited 2 to 10 fold increased lytic effect on melanoma and fibroblasts. Finally, nude mice harboring tumors made of a mix of melanoma cells and fibroblasts that were resistant to three administrations of Ad-F512 were treated intratumorally with five administrations of 1010 vp/mouse with either of the CRAds. Ad-F512 treatment induced the elimination of tumors in 2 out of 4 mice, while the other 2 mice showed a slight tumor growth. However in mice treated with Ad-NFkBSP(HRE)Pr we observed a complete elimination of the tumor in all mice that did not recur even after a follow up period of more than 100 days. These results indicate that addition of enhancer elements responsive to environmental conditions improved the therapeutic effect of a CRAd and might overcome the restriction imposed by the presence of stromal cells.