590. Efficient, Serum-Resistant Transfection of Murine Squamous Cell Carcinoma Cells by Metafectene and GeneJammer: Application to HSV-tk/Ganciclovir Gene Therapy
Authors: Krystyna Konopka, Nathan Overlid, Nejat Düzgünes
Journal: Molecular Therapy (2005)

Current treatments of head and neck squamous cell carcinoma (HNSCC) are largely unsatisfactory, and the five-year survival rate has not improved over the last two decades. The genetic approach to the treatment of HNSCC is based on the hypothesis that expression of therapeutic genes in target cells will cause a cytotoxic effect or mediate apoptosis. Oral cancer is a particularly appropriate target for gene therapy, since direct injection of genes into most primary and recurrent lesions is possible. Although generally efficient in transducing cells, viral vectors suffer from problems of immunogenicity, toxicity, limits in the size of exogenous DNA, and the risk of inducing tumorigenic mutations and generating active viral particles through recombination. Synthetic cationic liposome-DNA (lipoplexes) or cationic polymer-DNA complexes (polyplexes) constitute a promising alternative to the use of viral vectors and provide a simple means of transferring DNA into target cells. The inhibitory effect of serum on transfection mediated by non-viral vectors represents a serious limitation for their use in vivo. The presence of serum proteins, however, is unavoidable in vivo, and is advantageous in vitro because it increases cell viability and reduces transfection-associated cytotoxicity. In this study, we examined the effect of high concentrations of fetal bovine serum (FBS) on the delivery of luciferase, |[szlig]|-galactosidase and Herpes Simplex Virus thymidine kinase (HSV-tk) gene to murine squamous cell carcinoma cells, SCC-7, by the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer. Transfection was optimized using a luciferase-expressing plasmid. The level of gene expression in cell lysates was evaluated by measuring enzyme activity after a 48-h incubation. The optimal ratios for delivering the luciferase gene were 2 |[mu]|l Metafectene:1 |[mu]|g DNA and 3 |[mu]|l GeneJammer:0.5 |[mu]|g DNA. When transfection was performed in the presence of 20-60% FBS, both Metafectene- and GeneJammer-mediated luciferase expression were reduced only by about 20%. SCC-7 cells were also transfected with the |[szlig]|-galactosidase expressing plasmid in the absence or presence of 60% FBS. Approximately 60-70% of the cells were positive for |[szlig]|-gal staining. The expression of |[szlig]|-galactosidase was essentially not affected by serum. Cells transfected with the HSV-tk plasmid were incubated in the absence or the presence of ganciclovir (GCV; 20 |[mu]|g/ml) for the indicated periods of time. The Alamar Blue assay was used to determine GCV-mediated cytotoxicity. Mock-transfected cells served as controls. The delivery of the HSV-tk gene by Metafectene in the absence and presence of 60% FBS, followed by GCV treatment for 3 days, resulted in 68% and 36% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 60% cytotoxicity. After 6 days, 90-100% cytotoxicity was observed in the presence of 0% or 60% FBS. Our observations suggest that both GeneJammer and Metafectene may be useful for the gene therapy of OSCC in animal models.