DELIVERY OF HUMAN INTERFERON-GAMMA VIA GENE-TRANSFER IN-VITRO - PROLONGED EXPRESSION AND INDUCTION OF MACROPHAGE ANTIMICROBIAL ACTIVITY

Daily parenteral administration of exogenous interferon-gamma (IFN-gam ma) induces or accelerates recovery in experimental and human infectio ns, To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed, Th e complete coding region of IFN-gamma was amplified by RT-PCR and inse rted into an adenovirus cloning vector under the control of a human cy tomegalovirus promoter, Recombinant adenovirus containing the IFN-gamm a minigene (dAv-IFN-gamma) was isolated from 293 cells cotransfected w ith the linearized plasmid and an E1 region-deleted fragment of adenov irus genome, Following in vitro infection with dAv-IFN-gamma, dose-dep endent and time-dependent expression of IFN-gamma mRNA and production of soluble protein were demonstrated in human diploid fibroblast and H eLa cell cultures by Northern blot and ELISA, respectively, Extracellu lar protein secretion persisted for greater than or equal to 4 weeks f ollowing initial transfection, and secreted IFN-gamma induced both ant iviral activity (8000-25,000 U/ml) and macrophage activation with kill ing of intracellular Toxoplasma gondii and Leishmania donovani, These results establish that dAv-IFN-gamma generates long-term secretion of biologically active IFN-gamma in vitro and suggest that this vector ma y be a useful delivery system for cytokine therapy.