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:
VIALE DIEGO LUIS; LOPEZ MARIA VERONICA; CAFFERATA EDUARDO G; GOULD DAVID; CHERNAJOVSKY YUTI; PODHAJCER OSVALDO
Lugar:
Marriott Wardman Park Hotel, Washington, DC, USA
Reunión:
Congreso; 13th Annual Meeting of American Society of Gene and Cell Therapy; 2010
Institución organizadora:
American Society of Gene and Cell Therapy
Resumen:
Human tumors are composed of a heterogeneous mass of malignant cells that also include tumor-associated stromal cells that might become a barrier for successful therapies. 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 regulated 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 increase its replication and, therefore its oncolytic activity.We constructed twelve different chimeric promoters containing SPPr combined with Hypoxia Response Elements (HRE), NFκB-response elements (NFκB) or both and compared its transcriptional activity in luciferase expressing plasmids. Based on their activity/specificity ratio in different cell types we selected the chimeric promoter HRE-SPPr where three HREs were placed upstream of SPPr and a triple chimeric promoter NFκB-SP(HRE)Pr where twelve NFκB response elements were placed upstream of SPPr and 3 HREs inside SPPr sequence. We further evaluated the transcriptional activity of the chimeric promoters in an adenovirus backbone using luciferase as a reporter gene. We observed 2-10 fold induction of luciferase activity under hypoxia of both chimeric promoters and a 5-fold induction under TNFα treatment of NFκB-SP(HRE)Pr in malignant cells.Based on these results, we constructed two CRAds (Ad-HRE-SPPr and Ad-NFκB-SP(HRE)Pr) where E1A transcription was regulated by the chimeric promoters. Both CRAds exhibited 2 to 10 fold increased lytic effect under hypoxia on melanoma cells and fibroblasts compared to the parental Ad-F512.Finally, nude mice harboring tumors made of a mix of melanoma cells and fibroblasts that were resistant to three administrations of Ad-F512 (Lopez et al, PlosONE 2009) 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-NFκB-SP(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 tumor-environmental conditions improved the therapeutic effect of a CRAd and might overcome the restriction imposed by the presence of stromal cells.