Marked inhibition of glioblastoma target cell tumorigenicity in vitro by retrovirus-mediated transfer of a hairpin ribozyme against deletion-mutant epidermal growth factor receptor messenger RNA

Object. The goal of this study was to evaluate the activity of certain hair pin ribozymes against deletion-mutant epidermal growth factor receptor (Del ta EGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 80 1-bp deletion mutation associated with amplification of the EGFR gene is pr esent in a large subgroup of primary GBMs and confers enhanced tumorigenici ty in vivo. As a result of the deletion mutation, the fusion junction of th e gene is created directly upstream of a GTA tripler, which is subsequently transcribed into a ribozyme target codon (GUA). Methods. In attempts to intercept Delta EGFR gene expression at the mRNA le vel, the authors designed three different hairpin ribozymes derived from th e negative strands of satellite RNAs in tobacco ringspot virus, chicory yel low mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV 1, identified as the most active anti-Delta EGFR hairpin ribozyme motif, wa s cloned into the retroviral plasmid N2A+tRNA(i)(met). High-titer recombina nt retrovirus-containing supernatants (> 10(5) colony-forming units/ml) der ived from an amphotropic GP+envAM 12 packaging cell line transfected with t he N2A+rRNA(i)(met)-anti-Delta EGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.Delta EGFR glioblastoma cells, whi ch overexpress exogenous Delta EGFR. Using a virus/target cell ratio of 40: 1 in the absence of drug selection, the ribozyme transfer resulted in a gre ater than 90% reduction of Delta EGFR mRNA levels, a 69% inhibition of Delt a EGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions i n vitro: transfer of a control mutant ribozyme that was rendered incapable of cleaving its target yielded none of these effects. Conclusions. These findings indicate that the anti-Delta EGFR-sCYMV1 hairpi n ribozyme is capable of specifically inhibiting the expression of Delta EG FR and reversing the Delta EGFR-associated malignant phenotype of GEM cells . This strategy may constitute a promising gene therapy approach for a mole cularly defined subgroup of GBMs.