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。基因治疗是一种临床前和临床评价活跃的新方法。正在研究的策略包括使用不能复制的逆转录病毒或腺病毒（AD）递送自杀基因，如单纯疱疹病毒（HSV）胸苷激酶（tk）激活更昔洛韦（GCV），使其成为细胞毒性药物。宾夕法尼亚大学正在进行的一项恶性间皮瘤I期试验发现的一个主要限制是，在腔内分娩后，ADHSV tk进入肿瘤的渗透深度较差。克服这个问题的一个有希望的方法是使用具有复制能力的腺病毒。当这些病毒感染细胞并复制时，它会导致细胞裂解。此外，活性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的能力结合起来，我们假设抗肿瘤功效将得到增强。然而，这种具有复制能力的病毒的传递会引起一些安全问题。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。成功将导致临床基因治疗试验。基于FCCC最近发现的一个在人卵巢癌中具有特异性功能的启动子，本课题的目标是构建这样的载体，并在临床前评估其有效性和安全性，实现以下具体目标：开发和评估表达HSVtk的复制能力腺病毒载体。这将通过开发和测试含有HSVtk自杀基因的复制腺病毒载体来实现。在第一个系列的实验（原理证明）中，我们将研究一种包含HSVtk基因的完全复制病毒插入卵巢肿瘤模型的E3区域。这将使我们开始了解病毒复制与GCV传递的动力学。具体目标2。开发和评估一种表达HSVtk的具有复制能力的腺病毒载体，该载体只能在卵巢癌细胞中复制。这将通过开发Ad突变体来实现，该突变体使用Hamilton博士及其团队开发的“U3”启动子在卵巢肿瘤中选择性复制。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E1蛋白的早期产生，病毒复制将局限于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.