Ovarian selective Adenoviral vector for gene therapy of ovarian cancer

描述：(申请人描述)卵巢癌显然需要新的治疗方法。正在积极进行临床前和临床评估的一种新方法是基因治疗。正在研究的策略包括使用复制不能逆转录病毒或腺病毒(AD)来传递自杀基因，如单纯疱疹病毒(HSV)胸苷激酶(Tk)以激活更昔洛韦(GCV)成为细胞毒性药物。在宾夕法尼亚大学正在进行的恶性间皮瘤I期试验中发现的一个主要限制是腔内分娩后ADHSV tk对肿瘤的穿透深度较差。克服这一问题的一个有希望的方法是使用具有复制能力的腺病毒。当这种病毒感染细胞并复制时，它会导致细胞溶解。此外，活跃的病毒被释放出来感染其他肿瘤细胞。通过将这种增强杀伤和感染的机制与激活GCV的能力相结合，我们假设抗肿瘤效果将会增强。然而，这种复制能力强的病毒的交付引起了一些安全问题。为了解决这个问题，将构建一种在肿瘤细胞中有条件复制的病毒。成功将导致一项临床基因治疗试验。基于fccc最近发现的一种在人卵巢癌中具有功能特异性的启动子，本研究的目的是构建这类载体，并通过实现以下特定目的对其有效性和安全性进行临床前评估：1.建立和评价表达HSVtk的复制型腺病毒载体。这将通过开发和测试包含HSVtk自杀基因的复制腺病毒载体来实现。在第一系列实验中(原理证明)，我们将在卵巢肿瘤模型中研究一种完全复制的病毒，该病毒包含HSVtk基因插入E3区。这将使我们开始了解病毒复制与GCV传递的动态。特定目的2.开发和评价一种仅在卵巢癌细胞中复制的表达HSVtk的复制型腺病毒载体。这将通过开发利用汉密尔顿博士和他的团队开发的“U3”启动子在卵巢肿瘤中选择性复制的Ad突变体来实现。这些载体将通过破坏正常的Ad E1a启动子区域并将卵巢癌选择性启动子插入该区域而制成。由于复制依赖于早期产生的E1蛋白，病毒复制将限于那些肿瘤特异性启动子活跃的细胞。

DESCRIPTION: (Applicant's Description) New treatments for ovarian cancer are clearly needed. One novel approach under active preclinical and clinical evaluation is gene therapy. Strategies being investigated include use of replication incompetent retroviruses or adenoviruses (AD) to deliver suicide genes such as herpes simplex virus (HSV) thymidine kinase (tk) to activate ganciclovir (GCV) into a cytotoxic drug. One major limitation discovered in an ongoing phase I trial for malignant mesothelioma at the University of Pennsylvania is poor depth of penetration of ADHSV tk into the tumor after intracavitary delivery. A promising approach to overcome this problem is to use replication-competent adenoviruses. When such viruses infect cells and replicate, it causes cell lysis. In addition, active virus is released to infect other tumor cells. By coupling this mechanism of enhanced killing and infection with the ability to activate GCV, we hypothesize that anti-tumor efficacy will be enhanced. However, the delivery of such replication competent viruses causes some safety concerns. To address this issue, a virus conditionally replicative in tumor cells will be constructed. Success would result in a clinical gene therapy trial. Based on the recent discovery at FCCC of a promotor which shows specificity of function in human ovarian cancer, the goal of this proposal is to construct such vectors and preclinically evaluate their efficacy and safety by accomplishing the following specific aims: Specific Aim 1. Develop and evaluate a replication-competent adenoviral vector expressing HSVtk. This will be accomplished by developing and testing a replicating adenoviral vector containing the HSVtk suicide gene. In a first series of experiments (proof of principal), we will study a fully replicative virus containing the HSVtk gene inserted into the E3 region in ovarian tumor models. This will begin to allow us to understand the dynamics of viral replication vs delivery of GCV. Specific Aim 2. Develop and evaluate a replication-competent adenoviral vector expressing HSVtk that will only replicate in ovarian cancer cells. This will be accomplished by developing Ad mutants that replicate selectively in ovarian tumors using the "U3" promoter developed by Dr. Hamilton and his group. These vectors will be made by disrupting the normal Ad E1a promoter region and inserting the ovarian cancer-selective promoter into this region. Since replication is dependent on early production of E1 proteins, viral replication will be limited to those cells in which the tumor specific promoter is active.