Investigation of cellular localization of recombinant HSV-2 virions in infected vero cells for enhanced recovery

大多数工业生物过程寻求确保从其过程流中消除病毒。因此，大量工作投入到病毒清除研究中。然而，病毒本身可以是非常有用的产品，作为疫苗接种剂、肿瘤溶解疗法和基因传递载体。虽然有用，但在大规模应用程序中最大限度地恢复“活动”病毒方面，可用的信息很少。这项资助申请是为一名博士生和一名博士后进行的一项为期三年的研究，他们致力于捕获和纯化两种病毒：一种是伪型杆状病毒(一种改良的昆虫病毒)基因治疗载体，另一种是治疗性溶瘤马拉巴病毒，这种病毒在临床试验中显示出作为癌症治疗的巨大前景。这种捕获和提纯将使用膜技术来完成，这是Natrix分离公司核心业务的基础，并承诺将下游处理步骤的数量降至最低，并确保回收活跃的病毒。广泛的已知病毒及其独特的特性可能对制定强有力的普遍的病毒制剂下游战略--特别是那些将用于临床的病毒制剂--构成重大挑战。这些挑战包括病毒的数量、纯度和效力。为了尽量减少不同的特性，这项工作集中在水泡性口炎病毒囊膜糖蛋白G(VSV-G)伪型杆状病毒，以及VSV相关病毒，马拉巴病毒。许多基于病毒的载体用VSV-G进行伪分型，以增加它们的效力。将我们的净化策略建立在VSV-G的基础上，将允许使用Natrix技术平台来净化一些领先的候选治疗性病毒。两个主要战略将同时进行审查。首先，使用Natrix，将制作一种肝素功能化的膜，用于亲和捕获带有水泡性口炎样糖蛋白的病毒。其次，对Natrix离子交换膜进行评价。将对这两种策略在病毒恢复、纯度和效力方面的效果进行评估。

Most industrial bioprocesses seek to ensure the elimination of viruses from their process streams. As a result, a significant amount of work has gone into viral clearance research. However, viruses themselves can be very useful products as vaccination agents, oncolytic therapeutics and gene delivery vectors. Although useful, there is a dearth of information available on maximizing recovery of 'active' viruses for large scale applications. This grant application is for a three year study by a PhD student and a post-doctoral fellow on the capture and purification of two viruses: a pseudo-typed baculovirus (a modified insect virus) gene therapy vector, and a therapeutic oncolytic Maraba virus, which has shown tremendous promise in clinical trials as a treatment for cancer. This capture and purification will be done using membrane technologies, which form the basis of Natrix Separations Inc. core business, and that promise to minimize the number of downstream processing steps and ensure recovery of active virus. The wide array of known viruses and their distinct properties can pose major challenges in the development of robust universal downstream strategies for viral preparations - especially those destined for clinical use. These challenges include quantity, purity and potency of the viruses. To minimize the distinct properties, this work focuses on vesicular stomatitis virus envelope glycoprotein G (VSV-G) pseudo-typed baculovirus, and a VSV-related virus, the Maraba virus. Many virus-based vectors are pseudo-typed with VSV-G to increase their potency. Basing our purification strategies on the VSV-G, will allow a comprehensive strategy to purify a number of leading therapeutic viral candidates using the Natrix technology platform. Two major strategies will be examined in parallel. First, with Natrix, a heparin-functionalized membrane will be made for affinity capture of viruses with vesicular stomatitis-like glycoproteins. Second, Natrix ion exchange membranes will be evaluated. Both strategies will be evaluated for their effect on virus recovery, purity and potency.