Development of new therapy for brain tumors using recombinant oncolytic viruses

重组溶瘤性单纯疱疹病毒1型（HSV-1）载体是一种很有前景的脑肿瘤治疗剂。将治疗性转基因插入病毒基因组中，除了具有溶瘤活性外，还具有所需的抗肿瘤功能。由于溶瘤性HSV-1的疗效还取决于抗肿瘤免疫诱导的程度，因此免疫调节基因特别适合“武装”溶瘤性HSV-1载体。为了避免使用传统同源重组技术创建“武装”溶瘤性HSV-1载体所需的耗时过程，我们使用了利用细菌人工染色体和重组酶介导重组的创新构建系统。制备了表达免疫刺激基因的三基因缺失的HSV-1载体，并在携带低免疫原性神经2a（小鼠神经母细胞瘤）肿瘤的HSV-1易感AM小鼠中进行了测试。肿瘤内注射表达免疫刺激基因的“武装”溶瘤性HSV-1载体的效果明显高于未武装的对照HSV-1，并导致接种肿瘤和远端未接种肿瘤的生长抑制。对远处肿瘤的抗肿瘤作用不是由于病毒的扩散，而是由于诱导需要T淋巴细胞的全身抗肿瘤免疫。“武装”溶瘤性HSV-1载体在静脉注射时也能抑制皮下肿瘤的生长，而未武装的HSV-1则表现出最小的作用。结果表明，溶瘤性HSV-1治疗是一种有效的脑肿瘤治疗策略，用免疫刺激基因“武装”可以进一步提高溶瘤性HSV-1的疗效和有用性。

Recombinant oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for brain tumors. Insertion of therapeutic transgenes into the viral genome confers desired antitumor functions in addition to oncolytic activities. Because the efficacy of oncolytic HSV-1 also depends on the extent of antitumor immunity induction, immunomodulatory genes are particularly suited for "arming" oncolytic HSV-1 vectors. In order to circumvent time-consuming processes required with conventional homologous recombination techniques for creating "armed" oncolytic HSV-1 vectors, we used innovative construction systems utilizing bacterial artificial chromosome and recombinase-mediated recombinations. Triple gene-deleted HSV-1 vectors expressing immunostimulatory genes were generated and tested in HSV-1-susceptible AM mice bearing poorly-immunogenic Neuro2a (murine neuroblastoma) tumors. Intraneoplastic administration of "armed" oncolytic HSV-1 vectors expressing immunostimulatory genes resulted in significantly greater efficacy compared with unarmed control HSV-1, and led to growth inhibition of inoculated tumors as well as remote non-inoculated tumors. The antitumor effect on remote tumors was not due to viral spread but due to induction of systemic antitumor immunity requiring T lymphocytes. "Armed" oncolytic HSV-1 vectors could also suppress the growth of subcutaneous tumors when administered intravenously, whereas unarmed HSV-1 showed minimal effect. The results demonstrate that oncolytic HSV-1 therapy is a useful strategy for brain tumors, and "arming" with immunostimulatory genes can further improve the efficacy and usefulness of oncolytic HSV-1.