A NOVEL NONVIRAL CYTOPLASMIC GENE-EXPRESSION SYSTEM AND ITS IMPLICATIONS IN CANCER GENE-THERAPY

We recently have developed a unique cytoplasmic transient gene express ion system based on cotransfection of target cells with bacteriophage T7 RNA polymerase (RNAP) and plasmid DNA vectors containing a T7 autog ene. Because this T7 system is self-initiating, self-maintaining, and requires no cellular factors for transcription, it is therefore likely to function in any mammalian cell with any gene both in vitro and, mo re importantly, in vivo. In this study we demonstrate that the T7 DNA vector and T7 RNAP could be efficiently codelivered to cultured cells by lipofection. Different target genes were expressed by the T7 system in a wide variety of mammalian cells including several tumor cell lin es. Gene expression could be detected in more than 30% of the cells of some tumor cell lines transiently transfected by the T7 vector. Avera ge activity of the reporter enzyme (luciferase) expressed by a transfe cted cell was relatively constant regardless of the cell line used. Wh en a T7-luciferase vector was directly injected into various tissues o f mice without the use of liposomes, luciferase activity could be foun d in the injected liver, muscle, brain and tail connective tissues. Th e luciferase levels expressed by the T7 system were found to be up to 200-fold higher, depending upon the injected tissues, than levels achi eved with a traditional nuclear gene expression vector. Direct tumor i njection with a T7-beta-galactosidase (beta-gal) construct resulted in beta-gal gene expression in tumor cells near the injection sites. In addition, direct injection of the T7 system in mice did not generate d etectable quantities of antibodies against the T7 RNAP. These results suggest that this gene expression system may be useful in many differe nt medical applications such as cancer gene therapies and DNA vaccinat ion, where transient but rapid and efficient gene expression is requir ed.