624. Effect of an Oncolytic HSV Mutant Expressing ICP34.5 under Control of a Nestin Promoter for Brain Tumor Therapy

Abstract

HSV mutants (e.g. G207 and MGH1), possessing deletions in both copies of the ICP34.5 gene and an insertional mutation in the ICP6 gene (ribonucleotide reductase), have proven safe through a number of animal experiments and clinical trials. Although these double mutants clearly demonstrate oncolytic effects as well as anti-tumor immunization, their oncolytic effects are markedly reduced. ICP34.5-null viruses fail to grow in a variety of cultured cells due to their inability to prevent RNA-dependent protein kinase (PKR)-mediated inhibition of protein synthesis. In HSV-infected cells, viral double-stranded RNA will activate PKR by autophosphorylation, and activated PKR phosphorylates eIF-2a, which inhibits initiation of protein translation within the cells and in turn inhibits viral replication. The ICP34.5 dephosphorylates eIF-2a, thus allowing continued protein synthesis and viral replication. In order to increase therapeutic efficacy and tumor selectivity, glioma selective expression of ICP34.5 in oncolytic HSV has been explored. As a candidate for glioma selective promoters, we examined a synthetic nestin promoter, which consists of the nestin enhancer and hsp68 minimum promoter. We first performed RT-PCR to examine nestin expression in various types of cultured cells. All the glioma cell lines tested (U251, U87dEGFR, Gli36d5, T98G, MGH238, U138) were confirmed to be positive for nestin expression. Astrocytes, on the other hand, were negative. We, therefore, constructed a novel HSV mutant, rQNestin34.5, which expresses ICP34.5 under control of the synthetic nestin promoter. In the experiments using cultured human glioma cell lines in vitro, rQNestin34.5 showed remarkable enhancements in both viral propagation and oncolysis compared to the ICP34.5-null virus (rHsvQ1). In four primary glioblastoma cells, rQNestin34.5 also showed remarkable enhancement in both viral propagation and oncolysis compared to control rHsvQ1. In normal human astrocytes, rQNestin34.5 showed equivalent toxicity to control rHsvQ1. We also performed Western blotting to examine the phosphorylation state of eIF2a. In glioma cell lines infected with rQNestin34.5, the level of phspho-eIF2a was lower than that of control rHsvQ1, confirming that the ICP34.5 is expressed selectively in glioma cells and resulted in a decrease of phospho-eIF2a level. This was responsible for its efficient propagation in glioma cells. We further characterized the anti-cancer efficacy of this new virus in vivo using an athymic mouse subcutaneous tumor model (U87dEGFR cells) and a brain tumor model (U87dEGFR cells). The rQNestin34.5 showed significantly more potent inhibition of tumor growth compared to control rHsvQ1. The mice with brain tumor treated by rQNestin34.5 survived longer than those treated by rHsvQ1.