the Human SPARC Gene Inhibits Human. Melanoma Cell Growth In Vitro and In Vivo. Maria V. Lopez,1 Patricia V. Blanco,1 Diego L. Viale,1 Eduardo A.
CANCER-TARGETED GENE THERAPY: NON ADENOVIRAL TARGETING OF VECTORS AND NON VIRAL VECTORS demonstrated efficient transduction of human malignant glioma cells in culture. In the current investigation, we compared the transduction efficacy of LCMV GP- and vesicular stomatitis virus glycoprotein (VSV G)-pseudotyped lentiviral vectors for malignant glioma cells and normal brain cells in vitro and in vivo (Miletic et al., Human Gene Therapy 2004). LCMV pseudotypes transduced predominantly astrocytes, whereas VSV-G pseudotypes infected neurons as well as astrocytes in vitro and in the hippocampus and striatum of Fischer rats. LCMV pseudotypes showed an efficient transduction of solid glioma parts of 9L tumors (69.3 ± 10.6%) and a very specific transduction of infiltrating tumor cells (71.5 ± 10.6%). In contrast, VSV-G pseudotyped lentiviral vectors transduced only a few tumor cells in solid (6.8 ± 1.9%) and infiltrating (2.2 ± 1.9%) tumor parts and infected mostly normal brain cells in infiltrating tumor areas (42.9 ± 11.4%). To investigate the therapeutic impact of LCMV pseudotypes, we used the suicide gene HSV-tk fused to eGFP as therapeutic construct. We could demonstrate a high transduction efficiency of 9L tumors (Fig. 1A; Methionin-PET) using [18F]FHBG-PET to detect HSV-tk expression (Fig.1B) and by fluorescence microscopy of histological brain sections (Fig.1C). The Kaplan-Meier survival plot (Fig.1D) revealed over 90% of long time survivors (100 days) in the therapeutic group (n=13). In conclusion, lentiviral vectors pseudotyped with LCMV glycoproteins represent an attractive option for gene therapy of malignant glioma.
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78. 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 Maria V. Lopez,1 Patricia V. Blanco,1 Diego L. Viale,1 Eduardo A. Cafferata,1,2 David Gould,3 Yuti Chernajovsky,3 Osvaldo Podhajcer.1 1 Gene Therapy, Leloir Institute, Ciudad de Buenos Aires, Buenos Aires, Argentina; 2Genetica Experimental, Centro Nacional de Genetica Medica (ANLIS Carlos G Malbrán), Ciudad de Buenos Aires, Buenos Aires, Argentina; 3Bone and Joint Research Unit, University of London, London, United Kingdom. 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(∆10) that appears to be inhibitory, increased the promoter activity 4 to 7 fold in SPARCexpressing tumor cells and 3 to 5 fold in SPARC non-expressing cells. We investigated the properties of human SPARC promoterbased 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. The first two authors contributed equally.
Molecular Therapy Volume 11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
