Overproduction of the proinflammatory cytokines, tumor necrosis factor alpha (TNFa) and interleukin 1 (IL-1), contributes to the pathologic consequences of adult respiratory distress syndrome (ARDS), systemic inflammatory response syndrome and septic shock. However, current therapeutic approaches to deliver anticytokine therapies (IL-1ra, anti TNFa antibodies or anti TNF immunoadhesins) systemically are inherently inefficient and often ineffective. In particular, systemic administration of cytokine inhibitors at levels sufficient to neutralize exaggerated cytokine production in one organ may also block the presumably beneficial aspects of cytokine production in another. To develop an alternative approach for the targeted delivery of anticytokine therapies to organs or tissues adversely affected by acute inflammation, gene transfer of cytokine inhibitors or catalytic ribozymes directed against proinflammatory cytokine mRNA is proposed. Using cationic liposomes and mammalian expression plasmids, which results in only the transient expression (days to weeks) of foreign genes, the proposed studies will determine whether transgene expression of such human cytokine inhibitors as the extracellular domain of the p55 TNF receptor and IL-1ra, as well as the antiinflammatory cytokines, IL-10 and IL-4, can be directed to individual murine organs. In subsequent studies in mice and baboons, gene transfer will be employed to compare the efficacy of nonstable gene transfer on proinflammatory cytokine production (TNF, IL-1, IL-6 and KC), organ injury and outcome in several infection/organ injury models including endotoxin induced lung injury or cecal ligation and puncture (peritonitis). In addition, synthetically derived catalytic RNA sequences (hammerhead ribozymes) will be evaluated for their ability to cleave naked TNFa mRNA in vitro. Studies will then be conducted in vitro to determine whether delivery of plasmids containing ribozyme expressing genes can decrease TNFa levels and protein production in lipopolysaccharide stimulated peritoneal macrophages and RAW 264.7 murine macrophage cell lines. Finally, these expression plasmids containing DNA sequences for catalytic ribozymes will be administered to mice and baboons in an effort to reduce the exaggerated local tissue production of TNFa and IL-1B that is associated with organ damage.
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