Gene Therapeutics Research Institute, Cedars-Sinai Medical Center/UCLA, 8700 Beverly Blvd., Davis Building Research Pavilion, Room 5090, Los Angeles, CA 90048, USA.
J Virol. 2010 Jun;84(12):6007-17. doi: 10.1128/JVI.00398-10. Epub 2010 Apr 7.
Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Conditional cytotoxic/immune-stimulatory gene therapy (Ad-TK and Ad-Flt3L) elicits tumor regression and immunological memory in rodent GBM models. Since the majority of patients enrolled in clinical trials would exhibit adenovirus immunity, which could curtail transgene expression and therapeutic efficacy, we used high-capacity adenovirus vectors (HC-Ads) as a gene delivery platform. Herein, we describe for the first time a novel bicistronic HC-Ad driving constitutive expression of herpes simplex virus type 1 thymidine kinase (HSV1-TK) and inducible Tet-mediated expression of Flt3L within a single-vector platform. We achieved anti-GBM therapeutic efficacy with no overt toxicities using this bicistronic HC-Ad even in the presence of systemic Ad immunity. The bicistronic HC-Ad-TK/TetOn-Flt3L was delivered into intracranial gliomas in rats. Survival, vector biodistribution, neuropathology, systemic toxicity, and neurobehavioral deficits were assessed for up to 1 year posttreatment. Therapeutic efficacy was also assessed in animals preimmunized against Ads. We demonstrate therapeutic efficacy, with vector genomes being restricted to the brain injection site and an absence of overt toxicities. Importantly, antiadenoviral immunity did not inhibit therapeutic efficacy. These data represent the first report of a bicistronic vector platform driving the expression of two therapeutic transgenes, i.e., constitutive HSV1-TK and inducible Flt3L genes. Further, our data demonstrate no promoter interference and optimum gene delivery and expression from within this single-vector platform. Analysis of the efficacy, safety, and toxicity of this bicistronic HC-Ad vector in an animal model of GBM strongly supports further preclinical testing and downstream process development of HC-Ad-TK/TetOn-Flt3L for a future phase I clinical trial for GBM.
多形性胶质母细胞瘤（GBM）是一种致命的原发性脑肿瘤。条件性细胞毒性/免疫刺激基因治疗（Ad-TK 和 Ad-Flt3L）在啮齿动物 GBM 模型中引发肿瘤消退和免疫记忆。由于大多数入组临床试验的患者都会表现出腺病毒免疫，这可能会限制转基因的表达和治疗效果，因此我们使用高容量腺病毒载体（HC-Ads）作为基因传递平台。在此，我们首次描述了一种新型双顺反子 HC-Ad，可在单个载体平台中驱动单纯疱疹病毒 1 胸苷激酶（HSV1-TK）的组成型表达和 Tet 介导的 Flt3L 的诱导表达。即使存在全身 Ad 免疫，我们仍使用这种双顺反子 HC-Ad 实现了抗 GBM 的治疗效果，而没有明显的毒性。双顺反子 HC-Ad-TK/TetOn-Flt3L 被递送至大鼠颅内胶质瘤中。在治疗后长达 1 年的时间内，评估了生存、载体分布、神经病理学、全身毒性和神经行为缺陷。还在预先针对 Ads 免疫的动物中评估了治疗效果。我们证明了治疗效果，载体基因组局限于脑注射部位，并且没有明显的毒性。重要的是，抗腺病毒免疫并没有抑制治疗效果。这些数据代表了首个报告的双顺反子载体平台驱动两种治疗性转基因的表达，即组成型 HSV1-TK 和诱导型 Flt3L 基因。此外，我们的数据表明，在这个单载体平台内没有启动子干扰，并且可以最佳地进行基因传递和表达。在 GBM 动物模型中对这种双顺反子 HC-Ad 载体的疗效、安全性和毒性进行分析，强烈支持进一步进行临床前测试和下游工艺开发，以用于未来的 GBM 一期临床试验。
