Construction and Characterization of an Adenovirus Vector Containing a Bicistronic IL-12 Expression Cassette

Abstract

Gene therapy, although a young field, has become an intense area of study for the potential treatment of many inborn or acquired diseases, including cancer. The systemic or intratumoural delivery of cytokines and other immune system modulators has shown that protective, specific immunity to tumours is possible and effective. Adenovirus is an extremely useful vehicle for the transfer of genes, due to the ease of its preparation, its ability to be grown to high concentrations, and its capacity to infect a wide variety of cell types, including nonproliferating cells. Although a great deal of research has been done, there are a number of possible improvements, specifically, the consistency and range of transgene expression. The HCMV IE promoter/enhancer, used in a wide range of vector systems, has proven to drive lower levels of transgene expression in murine cells as compared to human cells. The use of the MCMV IE promoter/enhancer within the context of an adenovirus vector system has been investigated to see if more consistent transgene expression is possible in murine models. Studies with a luciferase reporter gene have demonstrated that in all cell types, the MCMV IE promoter/enhancer drives transgene expression to equivalent or higher levels than the HCMV IE promoter/enhancer. Our lab has investigated adenovirus as an intrtumoural gene delivery vehicle for various cytokines, including IL-12. However, the current IL-12-expressing vector carries the IL-12p35 subunit in E1 and the IL-12p40 subunit in E3. A homologous recombination event between E1 sequences in the viral genome and those that are integrated into 293 cells, would produce a replication-competent adenovirus expressing high levels of the p40 subunit, which is a potential antagonist to IL-12. A bicistronic adenovirus vector using the poliovirus IRES was constructed containing all IL-12 coding sequences in E1 to avoid this potential hazard. This virus expressed between 60 ng to 2.5 μg of IL-12/10⁶ cells, depending on the cell type. These levels of expression, however are 7-30-fold lower than those driven by previous IL-12 vector. Although this new virus is not appropriate for tumour immunotherapy, the shuttle plasmids developed during its construction have many potential uses.