Study of mechanism of action of cytokines and signaling molecules using adenoviral transient expression vectors

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Using adenoviral vetor (AdV) system, we have investigated the efficiency of gene expression, the effects of several AdV-mediated cytokine signals in vitro and in vivo, and the models of gene therapy.Infection of LacZ-Adv resulted in transient, dose-dependent expression in several cell lines and primary cells, showing that the order of strength of promoter activities was CAG>EF-1a>SRa. The expression levels of LacZ correlated well with the cell surface vitronectin receptor levels that are one of the cellular adenoviral receptors. The infection of several cytokine-expressing AdVs (GM-CSF,IL-2, IL-4, etc.) resulted in transient peaks of cytokine levels in vitro cultures (COS,HeLa) and in vivo injected mice. Trial of infection of AdV into lymphcytes was not successful, because lymphocytes express very low levels of vitronectin receptors and infection efficiency was very low. Infection at MOI>1000 resulted infection into only several % of cells with high cellular toxicity. Infection of GM-CSF-AdV into primary bone marrow cells resulted in factor-independent colony formation. We also injected AdVs without insert, or with GM-CSF,IL-4 cDNA,etc. into mice. Injection of very high titers of viruses resulted in death within a few days. Infection of GM-CSF vector resulted in enhanced hematopoiesis in spleen and bone marrow. We have also prepared several signaling molecule-expressing vectors, such as GM-CSF receptor a-and b-chains, their mutants, c-kit, c-fms, Jak kinase 1,2,3 and their dominant-negative mutants, and are now examining the effects of their expression on cellular growth and phenotypes.Based on the results of these three years, we can now obtain gross understanding of the efficiency and infection patterns of AdVs, and are continuing the studies of transferig signaling molecules into mouse disease models.