Reconstruction of an oncolytic HSV vector for systemic delivery

 产品说明：癌症病毒治疗是一种实用的方法，具有简单但实用的治疗机制-有目的地将病毒的内在溶细胞特性转化为恶性细胞的靶向杀伤。尽管最近在该领域已经取得了显著进展，但仍然存在的主要障碍是缺乏有效的手段来通过递送溶瘤病毒， 全身途径，这阻碍了其在治疗转移性疾病中的应用。影响溶瘤病毒通过全身途径递送效率的关键组分包括单核吞噬细胞系统（MPS）对病毒颗粒的吞噬、自然杀伤（NK）细胞对病毒感染的清除以及抗病毒抗体对病毒感染性的中和。此外，由于全身递送的病毒颗粒被大的bloo体积立即稀释，因此可能需要开发和应用可以主动富集病毒至肿瘤部位的递送策略。该项目旨在开发一系列新的策略来克服这些主要障碍;主要是，它将使用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，我们将测试我们的假设，即用“不要吃我”信号分子遗传包被FusOn-H2允许病毒在全身递送期间逃避MPS的清除。CD 47是一种主要的“不要吃我”信号分子。我们计划将CD 47的胞外结构域与病毒糖蛋白融合以包覆病毒，并将进行一系列体内实验以表明用CD 47基因包覆病毒确实可以增强针对转移性疾病的病毒疗法的全身递送。在目标2中，我们将测试嵌合分子HER 2-Col-Fc一旦掺入FusOn-H2中，就可以重定向NK细胞以攻击肿瘤细胞而不是清除病毒的想法。HER 2-Col-Fc包含三个关键结构域：肿瘤抗原HER 2特异性单链抗体、IgG Fc片段和胶原蛋白三聚化结构域。当以分泌形式表达时，该分子自身多聚化并以聚集体形式附着于表达HER 2的肿瘤细胞，然后可有效结合NK细胞上的低亲和力Fc受体（FcγRIIIA），以激活NK细胞从而杀死肿瘤细胞。这同时转移NK细胞清除溶瘤病毒。在目标3中，我们将证明一种新型细胞载体可以保护FusOn-H2，并通过全身途径将病毒主动递送到肿瘤部位。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，特异性靶向肿瘤新生血管。植入这种受体（T-eCAR）的T细胞可以特异性地附着并破坏肿瘤血管。我们将测试我们的假设，即T-eCAR可以作为全身递送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侵害，并主动将病毒有效载荷发送到肿瘤部位。虽然该项目主要旨在解决与溶瘤HSV的全身递送相关的障碍，但大多数策略可应用于其他溶瘤病毒或一般的基因递送载体。

 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.