EFFECTIVE REVERSAL OF A TRANSFORMED PHENOTYPE BY RETROVIRUS-MEDIATED TRANSFER OF A RIBOZYME DIRECTED AGAINST MUTANT N-RAS

A hammerhead ribozyme directed against oncogenic N-ras (N-13-ras) was introduced into a retroviral vector and its activity evaluated in vitr o and in cell lines. The catalytic efficiency of the ribozyme embedded within a 2618 nucleotides in vitro-generated transcript was not signi ficantly affected by the length of non-base pairing flanking sequences . A sensitive assay based on N-ras/luciferase fusion transcripts as a reporter system was used to assess ribozyme activity in mammalian cell s. More than 95% reduction in luciferase activity was observed in cell s transduced with a retrovirus containing the active form of the riboz yme, whereas no significant reduction was observed with the inactive f orm of the same ribozyme. in order to assay the activity of the retrov irally encoded ribozyme in a biological setting, the IL-3-dependent ce ll line TF-1 was transformed with N-13-ras. Expression of N-13-ras in these cells induced factor-independent colony growth and a dose-depend ent proliferative response to erythropoietin (Epo). Retrovirus-mediate d expression of the active form of the ribozyme in these cells restore d factor-dependent colony growth and abolished the proliferative respo nse to Epo. The reversion of the transformed phenotype correlated with a reduction in the amount of N-13-ras mRNA.