Characterization in vitro and in vivo of hammerhead ribozymes directed against murine tumor necrosis factor alpha

A hammerhead ribozyme directed against murine TNF alpha (mTNF alpha) mRNA h as been constructed. In vitro studies showed that this ribozyme was release d hom the parent molecule by Banking cis-acting hammerhead and hairpin ribo zymes. This same anti-mTNF alpha ribozyme specifically cleaved both synthet ically derived substrate RNA and mTNF alpha mRNA within a pool of total cel lular RNA. Endogenous delivery of this anti-mTNF alpha ribozyme via the sel f-cleaving cassette reduced mTNF alpha mRNA and protein levels in lipopolys accharide (LPS)-stimulated, stably transfected murine macrophage RAW 264.7 cells. When complexed to liposomes and exogenously delivered to mouse perit oneal macrophages, the same ribozyme, with and without the cis-acting riboz ymes, reduced mTNF alpha protein levels. However, an irrelevant ribozyme de livered in an identical fashion was also effective at reducing mTNF alpha p rotein levels. These data suggest that anti-mTNF alpha ribozymes can be con structed which efficiently cleave mTNF alpha mRNA, but irrelevant RNA/lipos ome complexes also effectively limit TNF alpha mRNA expression and can mimi c functional ribozyme activity under in vitro conditions. (C) 1999 Academic Press.