Abstract
To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIκB expressing both HSV-TK and a mutant form of the NF-κB inhibitor IκBα (IκBαM) was developed. TOIκB was constructed by recombining the IκBαM gene into the UL41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IκBαM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-κB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-κB was already translocated to the nucleus without receptor-dependent signaling by TNF-α. Following infection with TOIκB, nuclear translocation of NF-κB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIκB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIκB infection killed 85% of the cells compared to 20% killed by TOZ.1 infection. In the presence of GCV, these numbers increased to 95–100% for TOIκB and 80–85% for TOZ.1. TOIκB neurotoxicity measured on cultured murine neurons was relatively low and similar to that of TOZ.1. The survival of nude mice implanted into the brain with U-87MG tumor cells was markedly prolonged by intratumoral TOIκB injection and GCV administration. Survival of TOIκB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIκB only, PBS or PBS+GCV). These results suggest that IκBαM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.
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Acknowledgements
This work was supported by NIH Grant GM34534 to JCG, by GenVec, Inc. (Rockville, MD), and by a Grant-in-Aid for Scientific Research to SM (No. 12671357) from the Ministry of Education, Science and Culture, Ministry of Health and Human Welfare, Japan.
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Moriuchi, S., Glorioso, J., Maruno, M. et al. Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IκBα and HSV thymidine kinase. Cancer Gene Ther 12, 487–496 (2005). https://doi.org/10.1038/sj.cgt.7700816
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DOI: https://doi.org/10.1038/sj.cgt.7700816
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