SPECIFIC HAMMERHEAD RIBOZYME-MEDIATED CLEAVAGE OF MUTANT N-RAS MESSENGER-RNA IN-VITRO AND EX-VIVO - OLIGORIBONUCLEOTIDES AS THERAPEUTIC AGENTS

Two hammerhead ribozymes targeted to point mutations in codon 13 of th e N-ras oncogene were synthesized and their catalytic activity and sub strate specificity evaluated in vitro and ex vivo. In vitro studies sh owed that these ribozymes were specific for the oncogenic form of N-ra s, since cleavage was observed only in a 849-nucleotide-long transcrip t containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2'-modified to protect them against degra dation by nucleases. 2'-Fluoro-2'-deoxyuridine/cytidine-substituted ri bozymes were nearly as active as their unmodified counterparts, but ha d a prolonged stability in cell culture supernatant containing fetal c alf serum. The stability of the modified ribozymes increased by introd uction of terminal phosphorothioates groups without significant influe nce in their catalytic efficiency. A sensitive assay based on the use of N-ras/luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nea rly 60% in luciferase activity was observed in cells expressing mutant but not wild type N-ras/luciferase fusion transcripts. Moreover, clea vage of N-ras transcripts in HeLa cells was directly confirmed by a se mi-quantitative RT-PCR assay.