Use of nonviral in vivo jet-injection for hyperthermia-induced gene therapy

2090

Local gene transfer is an important strategy for efficient tumor targeted cancer gene therapy. Particularly for such local approaches physical technologies for nonviral gene transfer came into focus. Among those methods, nonviral jet-injection has developed to a feasible and reliable technique for the intratumoral transfer of naked plasmid-DNA. Jet-injection was used for the efficient in vivo transfer and expression of reporter and therapeutic genes. Here we show, that jet-injection is also applicable for the transfer of a heat-inducible vector into pre-established xenotransplanted human tumors in nude mice. This is an attractive approach, since employment of heat-inducible vector systems and hyperthermia treatment combines two treatment modalities for improved antitumoral efficacy. In our heat-inducible vector we use the proximal human multidrug resistance gene 1 (mdr1) promoter harboring heat-responsive elements, which is driving the expression of the human tumor necrosis factor alpha (TNF) gene. For the in vivo experiments we used pre-established HCT116 human colon carcinoma xenotransplanted in Ncr:nu/nu mice. At a size of 6x6mm tumors were either jet-injected with the heat-inducible vector or with the empty vector for the controls. Each animal received a single application of jet-injections representing a total DNA-dose of 0.04 mg per tumor. After jet-injection either kinetic studies of heat-induced TNF-expession or therapy experiments were started. The animals were treated with hyperthermia at 42°C for 60 minutes and tumors were removed 0, 4, 24 and 48 hours after heat-shock for quantitative real time PCR and ELISA to analyze the heat-induced TNF-expression. The expression analysis of the tumors revealed an heat-induced TNF-expression at the mRNA level and at the protein level. To evaluate the therapeutic efficacy of this nonviral approach, jet-injected HCT116 tumor harboring mice were treated with hyperthermia at 42°C for 60 minutes for induced TNF-expression, followed by adriamycin treatment (8.0 mg/kg i.v.) at days 1 and 8 after hyperthermia. This combined heat-induced TNF-expression and drug treatment led to significant tumor growth inhibition, which lasted for the entire observation time of 21 days. In contrast, animals which were only treated with hyperthermia or adriamycin did not show significant reduction in tumor growth. Therefore, this therapeutic nonviral approach is a demonstration, that in jet-injected tumors heat-induced TNF-expression and application of adriamycin is effective to achieve significant tumor growth inhibition