Cancer Gene Therapy by Direct Tumor Injections of a Nonviral T7 Vector Encoding a Thymidine Kinase Gene

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Abstract
Previously, we described a nonviral cytoplasmic gene therapy vector system based on the T7 autogene concept. This system has been shown to achieve rapid and high levels of gene expression in a variety of animal cells and tissues. To test the utility of the system in vivo tumor ablation, a T7 cancer gene therapy plasmid vector, pT7T7/T7TK, was constructed. This nonviral vector contains a T7 autogene, T7T7, and a human herpes simplex virus thymidine kinase (HSV-TK) gene driven by a second T7 promoter (T7TK). When co-transfected with T7 RNA polymerase (T7 RNAP) into cultured human osteosarcoma 143B cells, abut 10–20% of the cells were found to express HSV-TK, and more than 90% of the cells were killed in the presence of 1 μM ganciclovir (GCV) within 4 days after DNA transfection. The increase in killing above the transfection frequency is due to a “bystander” effect among transfected and untransfected 143B cells. Direct injections of pT7T7/T7TK into 143B tumors grown in nude mice resulted in TK gene expression in tumor cells located near the injection sites as revealed by the immunohistochemical staining. Repeated tumor injections of the pT7T7/T7TK vector and intraperitoneal (i.p.) injections of GCV resulted in inhibition of tumor growth and in tumor shrinkage in 6 out of 10 treated nude mice. Three of those six tumors fully regressed shortly after the end of the GCV injections. All of the full tumor regressions were found to be permanent and no apparent tumor relapses were observed for the rest of the lives of the treated nude mice after the initial tumor ablations. These results, combined with the nonviral and rapid cytoplasmic gene expression features, suggest that the T7 vector may be a good candidate for cancer gene therapy and other medical and biological applications. In short-term gene therapy applications such as cancer gene therapy, a nonviral vector that is capable of rapid and efficient therapeutic gene expression in the target tumor cells may be used in substitution of a viral vector. In this study, we demonstrate that repeated injections of a herpes simplex virus-thymidine kinase (HSV-TK) gene-encoding T7 nonviral vector delivered directly into solid 143B tumors grown in nude mice coupled with ganciclovir (GCV) i.p. injections resulted in tumor regressions.