INVESTIGADORES
VIALE Diego Luis
congresos y reuniones científicas
Título:
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
Autor/es:
LOPEZ, MV; BLANCO, P; VIALE, DL; CAFFERATA, EG; GOULD D; CHERNAJOVSKY Y; PODHAJCER, OL
Lugar:
America's Center, St. Louis, MO, USA
Reunión:
Congreso; Eighth Annual Meeting of the American Society of Gene Therapy; 2005
Institución organizadora:
American Society of Gene Therapy
Resumen:
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(|[Auml]|10) 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.