Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras

Abstract

A hammerhead ribozyme directed against oncogenic N-ras (N13-ras) was introduced into a retroviral vector and its activity evaluated in vitro and in cell lines. The catalytic efficiency of the ribozyme embedded within a 2618 nucleotides in vitro-generated transcript was not significantly 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 cells. More than 95% reduction in luciferase activity was observed in cells transduced with a retrovirus containing the active form of the ribozyme, whereas no significant reduction was observed with the inactive form of observed with the inactive form of the same ribozyme. In order to assay the activity of the retrovirally encoded ribozyme in a biological setting, the IL-3-dependent cell line TF-1 was transformed with N13-ras. Expression of N13-ras in these cells induced factor-independent colony growth and a dose-dependent proliferative response to erythropoietin (Epo). Retrovirus-mediated expression of the active form of the ribozyme in these cells restored factor-dependent colony growth and abolished the proliferative response to Epo. The reversion of the transformed phenotype correlated with a reduction in the amount of N13-ras mRNA.