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.
OBJECTIVE
The goal of this study was to evaluate the activity of certain hairpin ribozymes against deletion-mutant epidermal growth factor receptor (deltaEGFR) messenger (m)RNA in glioblastomas multiforme (GBMs). A distinct 801-bp deletion mutation associated with amplification of the EGFR gene is present in a large subgroup of primary GBMs and confers enhanced tumorigenicity in vivo. As a result of the deletion mutation, the fusion junction of the gene is created directly upstream of a GTA triplet, which is subsequently transcribed into a ribozyme target codon (GUA).


METHODS
In attempts to intercept deltaEGFR gene expression at the mRNA level, the authors designed three different hairpin ribozymes derived from the negative strands of satellite RNAs in tobacco ringspot virus, chicory yellow mottle virus (sCYMV1), and arabis mosaic virus against this target and evaluated their efficiency and specificity in a cell-free system. The sCYMV1, identified as the most active anti-deltaEGFR hairpin ribozyme motif, was cloned into the retroviral plasmid N2A+tRNAi(met). High-titer recombinant retrovirus-containing supernatants (> 10(5) colony-forming units/ml) derived from an amphotropic GP+envAM 12 packaging cell line transfected with the N2A+tRNAi(met)-anti-deltaEGFR-sCYMV1 construct were used to introduce the sCYMV1 hairpin ribozyme into U-87MG.deltaEGFR glioblastoma cells, which overexpress exogenous deltaEGFR. Using a virus/target cell ratio of 40:1 in the absence of drug selection, the ribozyme transfer resulted in a greater than 90% reduction of deltaEGFR mRNA levels, a 69% inhibition of deltaEGFR-mediated proliferation advantage, and a greater than 95% decrease of colony formation in soft agar under relative serum starvation conditions in 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-deltaEGFR-sCYMV1 hairpin ribozyme is capable of specifically inhibiting the expression of deltaEGFR and reversing the deltaEGFR-associated malignant phenotype of GBM cells. This strategy may constitute a promising gene therapy approach for a molecularly defined subgroup of GBMs.
Halatsch
M E
ME
Department of Medicine, The Mount Sinai School of Medicine, New York, New York, USA.
Schmidt
U
U
Bötefür
I C
IC
Holland
J F
JF
Ohnuma
T
T
D001483:Base Sequence; D001932:Brain Neoplasms / Q000235:genetics* / Q000628:therapy; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D002872:Chromosome Deletion; D004252:DNA Mutational Analysis; D066246:ErbB Receptors / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005909:Glioblastoma / Q000235:genetics* / Q000628:therapy; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics; D016133:Polymerase Chain Reaction; D016337:RNA, Catalytic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014410:Tumor Stem Cell Assay
D016337:RNA, Catalytic; D012333:RNA, Messenger; D066246:ErbB Receptors