Particle-mediated (gene gun) in vivo delivery of the murine interleukin 12 (IL-12) gene in an expression plasmid was evaluated for antitumor activity. Transfer of IL-12 cDNA into epidermal cells overlying an implanted intradermal tumor resulted in detectable levels (266.0 ± 27.8 pg) of the transgenic protein at the skin tissue treatment site. Despite these low levels of transgenic IL-12, complete regression of established tumors (0.4-0.8 cm in diameter) was achieved in mice bearing Renca, MethA, SA-1, or L5178Y syngeneic tumors. Only one to four treatments with cDNA-coated particles, starting on day 7 after tumor cell implantation, were required to achieve complete tumor regression. This antitumor effect was CD8+ T cell-dependent and led to the generation of tumor-specific immunological memory. By using a metastatic P815 tumor model, we further showed that a delivery of IL-12 cDNA into the skin overlying an advanced intradermal tumor, followed by tumor excision and three additional (5, 6). However, systemic administration of rIL-12 caused dose-dependent toxicity in mice (7) and in human trials (8). Thus, a delivery mechanism that can provide relatively low levels of IL-12 at the target tissue might be advantageous in that it could generate an antitumor effect without causing systemic toxicity. Indeed, as cancer gene therapy has evolved, recent studies have produced encouraging results, showing that murine fibroblasts (9) or tumor cells (10) transduced in vitro with the IL-12 gene using a retroviral vector were able to induce antitumor immune responses. These data suggest that peritumoral IL-12 delivery may be as efficacious as systemic administration and avoid many undesirable side effects.The particle-mediated method for gene delivery by gene gun utilizes a shock wave to accelerate DNA-coated gold particles into target cells or tissues. At submicrogram quantities of DNA per dose for in vitro or in vivo gene transfer, the gene gun can deliver thousands of DNA copies intracellularly into test tissues, resulting in high level transgene expression (11). As this method is cell surface receptor-independent, it can successfully deliver genes into a wide spectrum of mammalian cell types (12, 13). We have recently demonstrated that a particlemediated, in vivo cytokine gene therapy reduces tumor growth in mice (14). Treatments with interferon-y and tumor necrosis factor a shortly after the implantation of tumor cells inhibited tumor growth and prolonged the survival of tumor-bearing mice. To more closely approximate clinical situations, and to take advantage of the findings that IL-12 more effectively stimulates activated than naive T cells (15, 16), we evaluated the effect of IL-12 gene therapy on the growth of established tumors. In this study, we utilized the gene gun technology for in vivo IL-12 cDNA delivery into the skin overlying the implanted, established tumor tissues. MATERIALS AND METHODSMice. BALB/c, C57BL/6, DBA/2, and A/J female mice between 8 and 12 weeks of age were obtained from HarlanSparagu...

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Implantation of tumor cells modified by in vitro cytokine gene transfer has been shown by many investigators to result in potent in vivo antitumor activities in mice. Here we describe an approach to tumor immunotherapy utilizing direct transfection of cytokine genes into tumorbearing animals by particle-mediated gene transfer. In vivo transfection of the human interleukin 6 gene into the tumor site reduced methylcholanthrene-induced fibrosarcoma growth, and a combination of murine tumor necrosis factor a and interferon y genes inhibited growth of a renal carcinoma tumor model (Renca). In addition, treatment with murine interleukin 2 and interferon -y genes prolonged the survival of Renca tumor-bearing mice and resulted in tumor eradication in 25% of the test animals. Transgene expression was demonstrated in treated tissues by ELISA and immunohistochemical analysis. Significant serum levels of interleukin 6 and interferon y were detected, demonstrating effective secretion of transgenic proteins from treated skin into the bloodstream. This in vivo cytokine gene therapy approach provides a system for evaluating the antitumor properties ofvarious cytokines in different tumor models and has potential utility for human cancer gene therapy.