Combination gene therapy for glioblastoma involving herpes simplex virus vector-mediated codelivery of mutant IkappaBalpha and HSV thymidine kinase.
To improve the effectiveness of herpes simplex virus (HSV) thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene therapy, the replication-defective HSV vector TOIkappaB expressing both HSV-TK and a mutant form of the NF-kappaB inhibitor IkappaBalpha (IkappaBalphaM) was developed. TOIkappaB was constructed by recombining the IkappaBalphaM gene into the U(L)41 locus of a replication-defective lacZ expression vector, TOZ.1. Expression of IkappaBalphaM was confirmed by Western blotting, and the ability of the mutant protein to inhibit NF-kappaB nuclear translocation was examined by electrophoretic mobility shift assay. In human glioblastoma U-87MG cells, the p50/p50 dimer of NF-kappaB was already translocated to the nucleus without receptor-dependent signaling by TNF-alpha. Following infection with TOIkappaB, nuclear translocation of NF-kappaB in U-87MG cells was significantly inhibited and caspase-3 activity increased compared with TOZ.1-infected cells. The cytotoxicity of TOIkappaB for U-87MG cells was investigated by colorimetric MTT assay. At an MOI of 3, TOIkappaB 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 TOIkappaB and 80-85% for TOZ.1. TOIkappaB 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 TOIkappaB injection and GCV administration. Survival of TOIkappaB+GCV group was significantly longer (P<.02, Wilcoxon test) than for the control groups (TOZ.1 or TOIkappaB only, PBS or PBS+GCV). These results suggest that IkappaBalphaM expression may be a safe enhancement of replication-defective HSV-based suicide gene therapy in vitro and in vivo.
Moriuchi
Shusuke
S
Department of Neurosurgery, Osaka Graduate University Medical School, 2-2 Yamada-oka, Suita city, Osaka 565, Japan. moriuchi@onh.go.jp
Glorioso
Joseph C
JC
Maruno
Motohiko
M
Izumoto
Shuichi
S
Wolfe
Darren
D
Huang
Shaohua
S
Cohen
Justus B
JB
Yoshimine
Toshiki
T
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000166:cytology; D001932:Brain Neoplasms / Q000628:therapy*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003131:Combined Modality Therapy; D005260:Female; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D005822:Genetic Vectors; D005909:Glioblastoma / Q000628:therapy*; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D010957:Plasmids / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics* / Q000378:metabolism; D014407:Tumor Cells, Cultured
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D000072000:NF-KappaB Inhibitor alpha; D013937:Thymidine Kinase; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases