Reconstruction of an oncolytic HSV vector for systemic delivery

 描述：癌症病毒疗法是一种实用的方法，具有简单而实用的治疗机制--有目的地将病毒固有的细胞溶解特性转化为对恶性细胞的靶向杀伤。尽管最近在这一领域取得了重大进展，但仍然存在的一个主要障碍是缺乏有效的手段通过以下方式传递溶瘤病毒 全身途径，这阻碍了它在治疗转移性疾病中的应用。通过全身途径影响溶瘤病毒传递效率的关键成分包括单核吞噬细胞系统(MPS)吞噬病毒颗粒，自然杀伤(NK)细胞清除病毒感染，以及抗病毒抗体中和病毒感染性。此外，由于系统传递的病毒颗粒会立即被大量出血稀释，因此可能需要开发和应用一种能够主动将病毒浓缩到肿瘤部位的传递策略。该项目旨在开发一系列新的策略来克服这些主要障碍；首先，它将使用一种最初在我们实验室构建的独特的溶瘤病毒(Fuson-H2)。在目标1中，我们将检验我们的假设，即在Fuson-H2上遗传地覆盖一种“不要吃我”的信号分子，使病毒在全身递送期间逃脱MPS的清除。CD47是一个主要的“不要吃我”的信号分子。我们计划将CD47的胞外区与病毒糖蛋白融合在一起，以覆盖病毒，并将进行一系列体内实验，以证明用CD47基因覆盖病毒确实可以增强针对转移性疾病的病毒治疗的系统递送。在目标2中，我们将测试嵌合分子HER2-Col-Fc一旦被整合到Fuson-H2中，就可以重定向NK细胞攻击肿瘤细胞，而不是清除病毒。HER2-Col-Fc包含三个关键区：针对肿瘤抗原HER2的单链抗体、来自免疫球蛋白的Fc片段和来自胶原的三聚结构域。当以分泌形式表达时，该分子会自我聚合并以聚集体的形式附着在表达hER2的肿瘤细胞上，这些聚集体可以有效地与NK细胞上的低亲和力Fc受体(FcγRIIIA)结合，激活它们从而杀伤肿瘤细胞。这同时转移了NK细胞清除溶瘤病毒的能力。在目标3中，我们将展示一种新型的细胞载体可以保护Fuson-H2，并通过全身途径主动将病毒运送到肿瘤部位。在我们最近的研究中，我们已经产生了一种新的嵌合抗原受体，它特异性地针对肿瘤新生血管。植入这种受体的T细胞(T-eCar)可以特异性地附着和破坏肿瘤血管。我们将验证我们的假设，即T-eCar可以作为系统传递Fuson-H2的完美细胞载体，因为它们可以保护病毒免受中和抗体的攻击，并主动将病毒有效载荷发送到肿瘤部位。虽然这个项目主要是为了解决与系统性输送溶瘤单纯疱疹病毒相关的障碍，但大多数策略可以应用于其他溶瘤病毒或一般的基因输送载体。

 DESCRIPTION: Cancer virotherapy is a pragmatic approach with a simple, yet practical, therapeutic mechanism - purposeful conversion of the intrinsic cytolytic property of a virus into targeted killing of malignant cells. Although significant progress has recently been made in this field, a remaining major obstacle is the lack of an efficient means to deliver oncolytic viruses by the systemic route, which is hampering its application in treating metastatic diseases. Key components that affect the delivery efficiency of oncolytic viruses by the systemic route include engulfment of viral particles by the mononuclear phagocyte system (MPS), clearance of virus infection by natural killer (NK) cells, and neutralization of virus infectivity by antiviral antiboies. Additionally, as the systemically delivered viral particles are instantly diluted by the large bloo volume, a delivery strategy that can actively enrich viruses to the tumor site may need to be developed and applied. This project is purposefully designed to develop a series of novel strategies to overcome these major obstacles; primarily, it will use a unique oncolytic virus (FusOn-H2) that was originally constructed in our lab. In aim 1, we will test our hypothesis that genetically coating FusOn-H2 with a "don't eat me" signal molecule allows the virus to escape the clearance of MPS during systemic delivery. CD47 is a major "don't eat me" signal molecule. We plan to fuse the extracellular domain of CD47 with a viral glycoprotein to coat the virus and will conduct a series of in vivo experiments to show that genetically coating the virus with CD47 can indeed enhance systemic delivery of virotherapy against metastatic disease. In aim 2, we will test the idea that a chimeric molecule, HER2-Col-Fc, once incorporated into FusOn-H2, can redirect NK cells to attack tumor cells instead of clearing the virus. HER2-Col-Fc contains three key domains: the single chain antibody specific for the tumor antigen HER2, the Fc fragment from IgG and the trimerization domain from collagen. When expressed as a secreted form, this molecule self multimerizes and attaches to HER2-expressing tumor cells as aggregates, which can then efficiently bind to the low affinity Fc receptor (FcγRIIIA) on NK cells to activate them o kill tumor cells. This simultaneously diverts NK cells from clearing oncolytic virus. In aim 3, we will demonstrate that a novel cell carrier can protect FusOn-H2 as well as actively deliver the virus to tumor sites by the systemic route. In our recent studies, we have generated a novel chimeric antigen receptor that specifically targets tumor neovasculature. T cells engrafted with this receptor (T-eCAR) can specifically attach to and destroy tumor blood vessels. We will test our hypothesis that T-eCAR can function as a perfect cell carrier for systemic delivery of FusOn-H2, as they can protect the virus from the neutralizing antibodies and actively send the virus payload to the tumor site. Although this project is mainly designed to address the obstacles associated with systemic delivery of an oncolytic HSV, most of the strategies can be applied to other oncolytic viruses or to gene delivery vectors in general.