Gene gun-mediated skin transfection with interleukin 12 gene results in regression of established primary and metastatic murine tumors.

Abstract

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...

Molecular strategies for improving cytokine transgene expression in normal and malignant tissues

“…These cytokines were repeatedly shown to exhibit functional biological activity, as many researchers have previously reported. 22,29,30 A comparison of the results from our current study versus those reported by Rajagopalan and Malter 22 suggests that 3 0 cDNA structural features may influence transgenic protein expression via additional mechanisms besides mRNA stability and polysome-run-on efficiency. It is currently unclear to us as to which mechanism(s) (eg, RNA stability) is playing the key role for enhanced cytokine gene expression, and additional experiments are needed to demonstrate the possible post-transcriptional, translational or other processing regulations of these transgenic RNA or DNA vector systems.…”

“…The quantity of transgenic murine IL-12 protein heterodimer was determined by a cell proliferation bioassay using ConA-activated splenocytes, as previously described. 30 Human IL-12 levels were determined via a p35/p40 heterodimer-specific ELISA kit (R&D Systems, Minneapolis, MN, USA). Based on results from this study, we estimate that an increase of at least seven to 30-fold in various cytokine transgene expression levels could be readily obtained by employing a combination of such molecular strategies for vector, promoter or other sequence constructions.…”

“…This was demonstrated by their capacity to elicit CTL or histological inflammatory activity and potent antitumor activities effectively. 12,13,29,[30][31][32] Together with the findings of the relative capacities of different cDNA constructs in the current study, we believe that these results can be usefully employed by researchers for the proper and specified use of 3 0 UTR deletion for cytokine expression vectors in a clinically relevant study, that is, different as well as the same cytokine species can differ drastically in transgene expression under different in vivo and ex vitro conditions, and hence specific cytokine expression vectors do need to be carefully tested in target or related cells/tissues before clinical studies.…”

“…In previous studies on cytokine gene expression, it has been documented that mutating the AU-rich sequences of mRNA in the 3 0 UTR can effectively enhance mRNA stability, resulting in an augmented transgenic cytokine protein expression [18][19][20]22,30 These results were accomplished via an overlapping PCR technique, in which the AUUUA regions of the 3 0 UTR were replaced with AUGUA pentamer repeats. 20,22 There were also suggestions that a group of cytokine gene constructs, with the 3 0 UTR totally removed by cDNA cloning, expressed very well in transgenic studies.…”

Adenovirus-mediated interleukin-12 gene therapy for prostate cancer: suppression of orthotopic tumor growth and pre-established lung metastases in an orthotopic model

“…These include injection of genetically engineered fibroblasts for the secretion of IL-12 in an orthotopic melanoma model; direct transfer of IL-12 cDNA via gene gun-mediated transfection of skin tissue overlaying subcutaneous melanomas; direct intratumoral injection of adenovirus expressing IL-12 in ectopic breast and colon cancer models; and intradermal injection of IL-12 cDNA and vaccination using irradiated IL-12-transfected tumor cells in a metastatic colon cancer model. [15][16][17][18][19][20][21] These models suggest that IL-12 gene therapy results in tumor regression and suppression of metastasis.…”