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 observed with the inactive form of the same ribozyme. In (N 13 -ras) was introduced into a retroviral vector and its order to assay the activity of the retrovirally encoded activity evaluated in vitro and in cell lines. The catalytic ribozyme in a biological setting, the IL-3-dependent cell line efficiency of the ribozyme embedded within a 2618 nucleo-TF-1 was transformed with N 13 -ras. Expression of N 13 -ras tides in vitro-generated transcript was not significantly in these cells induced factor-independent colony growth affected by the length of non-base pairing flanking and a dose-dependent proliferative response to erythroposequences. A sensitive assay based on N-ras/luciferase ietin (Epo). Retrovirus-mediated expression of the active fusion transcripts as a reporter system was used to assess form of the ribozyme in these cells restored factor-depenribozyme activity in mammalian cells. More than 95% dent colony growth and abolished the proliferative reduction in luciferase activity was observed in cells transresponse to Epo. The reversion of the transformed phenoduced with a retrovirus containing the active form of type correlated with a reduction in the amount of N 13 -ras the ribozyme, whereas no significant reduction was mRNA.

A sensitive dual-fluorescence reporter system enables positive selection of ras suppressors by suppression of ras-induced apoptosis

“…pL9RFP was made by insertion of the Not1/Sal1 fragment of RFP cDNA excised from pDsRed-N1 (Clontech, Palo Alto, CA) into pL9XL. The hammerhead ribozyme MRE763C designed to specifically cleave the N-ras m transcript 14,15 was synthesized as two complementary synthetic oligonucleotides carrying linkers with BamH1 and Not1 restriction sites at the 5 0 and 3 0 ends, respectively. This ribozyme was inserted into pL9XL to generate L9Rz.…”

“…To achieve this, one reporter (GFP) was coexpressed with N-ras m 22 and the other reporter (RFP) 23,24 was coexpressed with a ribozyme previously shown to specifically suppress N-ras m . 14,15 We show that ribozyme-mediated ras suppression inhibits growth in NIH3T3 and TF-1 cells, but produces a growth advantage in K562 cells since N-ras m expression inhibits growth in the K562 cells. Similar effects were produced by two other inhibitors of Ras signaling, namely the MEK 1/2 and PI3 kinase inhibitors.…”

“…In the existing models, monitoring of Ras suppression and particularly the identification of candidate ras suppressors is problematic. A major obstacle in assessing Ras suppressor activity is that positive activity results in loss of cell growth and viability, 15,[17][18][19] which renders them difficult to identify.…”

“…Antisense and ribozyme targeting of mutant Ras genes have both been shown to produce antitumor activity in model systems. [14][15][16][17][18][19] A variety of agents are being developed, some of which have moved into clinical trials. These agents tend to inactivate downstream effectors of the Ras signal cascade, particularly the protein kinases.…”

Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta

“…These ribozymes have been applied to downregulate a variety of genes, and their potential as therapeutic agents in the management of genetic disorders, particularly those whose pathogenesis is the result of a dominant-negative effect exerted by a mutant gene product, has been widely recognized. [8][9][10][11] More recently, the discovery of the role of RNA interference (RNAi) in post-transcriptional gene silencing has led to the suggestion that a new class of RNA-based therapeutics might be developed. [12][13][14] Unlike the antisense and RNAi approaches, which both rely on the action of the cellular machinery for their effect, hammerhead ribozymes act directly to cleave their target mRNA.…”