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-gamma) induces or accelerates recovery in experimental and human infections. To develop an alternative delivery system, a replication-defective recombinant adenovirus expressing human IFN-gamma was constructed. The complete coding region of IFN-gamma was amplified by RT-PCR and inserted into an adenovirus cloning vector under the control of a human cytomegalovirus promoter. Recombinant adenovirus containing the IFN-gamma minigene (dAv-IFN-gamma) was isolated from 293 cells co-transfected with the linearized plasmid and an E1 region-deleted fragment of adenovirus genome. Following in vitro infection with dAv-IFN-gamma, dose-dependent and time-dependent expression of IFN-gamma, mRNA and production of soluble protein were demonstrated in human diploid fibroblat and HeLa cell cultures by Northern blot and ELISA, respectively. Extracellular protein secretion persisted for > = 4 weeks following initial transfection, and secreted IFN-gamma induced both antiviral activity (8000-25,000 U/ml) and macrophage activation with killing 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 may be a useful delivery system for cytokine therapy.
Stoeckle
M Y
MY
Department of Medicine, Cornell University Medical College, New York, NY, USA.
Falck-Pederson
E
E
Rubin
B Y
BY
Anderson
S L
SL
Murray
H W
HW
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D007893:Leishmania donovani / Q000276:immunology; D008262:Macrophage Activation; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D014122:Toxoplasma / Q000276:immunology
D011994:Recombinant Proteins; D007371:Interferon-gamma