Expression of the Herpes Simplex Virus Thymidine Kinase gene by a promoter region of the human SPARC gene inhibits human melanoma cell growth in vitro and in vivo

M. VERONICA LOPEZ; PATRICIA BLANCO; DIEGO VIALE; EDUARDO A. CAFFERATA; DAVID GOULD; YUTI CHERNAJOVSKY; OSVALDO L. PODHAJCER

Saint Louis. USA

Congreso; 8th Annual Meeting of the American Society of Gene Therapy; 2005

American Society of Gene Therapy

Transcriptional targeting utilizes tumor-associated gene (TAG) promoters to direct the expression of therapeutic genes specifically to the malignant tumor cells. However, solid human tumors are highly heterogeneous and TAGs are currently expressed in only a percentage of malignant cells. In addition, tumor progression evolves as a cross talk between the different cell types within the tumor and the surrounding stroma, endothelial cells and fibroblasts. As a first approach to co-target the different components of a tumor mass we investigated the properties of the human SPARC promoter. SPARC expression is highest during embryo development in bone and cartilage areas while in adults its expression is restricted to tissues undergoing extensive cellular turnover. Different studies have shown that SPARC is overexpressed in a variety of cancer types not only in malignant cells but also in tumor-associated fibroblasts and endothelium. First, we compared the transcriptional activity of different constructs encompassing the human SPARC promoter region to drive luciferase expression in human malignant cells from different origins as well as in fibroblasts and endothelial cells. We analyzed the activity of a fragment corresponding to the SPARC promoter (hSPPr) that extends from -1176 bp to +71 bp and includes the untranslated exon 1. hSPPr activity was higher in malignant cells, fibroblasts and endothelial cells overexpressing SPARC compared to cells with lower levels or none SPARC expression. A SPARC promoter version with a 10 bp deletion between two GGA boxes hSPPr(D10) that appears to be inhibitory, increased the promoter activity 4 to 7 fold in SPARC-expressing tumor cells and 3 to 5 fold in SPARC non-expressing cells. We investigated the properties of human SPARC promoter-based gene therapy. We prepared plasmid-based vectors carrying the thymidine kinase gene (TK) driven by the different forms of SPARC promoter and evaluated its efficacy in the presence of gancyclovir (GCV) in vitro, in cell culture and spheroids, and in vivo after xenograft transplantation. SPARC-expressing melanoma cells stably producing TK driven by hSPPr (MEL-SPPr-TK) were killed in the presence of GCV when grown as monolayers and spheroids even when the latter were made of malignant cells and fibroblasts. The sensitization to GCV was retained in vivo. Indeed, almost complete tumor regression was observed in nude mice injected with MEL-SPPr-TK cells and treated daily with GCV starting from day 10 after tumor cells injection. Thus, suicide gene therapy driven by SPARC gene promoter appears as a useful strategy for conditional targeting in cancer gene therapy. ±Supported by the Welcome Trust, UK, The National Agency for Promotion of Science and Technology, René Barón Foundation and AFULIC, Argentina. *The first two authors contributed equally Keywords: Cancer Gene Therapy, Targeted Gene Expression, SPARC