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

大多数工业生物工艺都力求确保从其工艺流程中消除病毒。因此，大量的工作已经进入病毒清除研究。然而，病毒本身可以是非常有用的产品，如疫苗接种剂、溶瘤疗法和基因传递载体。虽然有用，但缺乏关于如何最大限度地恢复大规模应用程序的“活动”病毒的可用信息。这项拨款申请是为一名博士生和一名博士后进行为期三年的研究，目的是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病毒马拉巴病毒，这两种病毒在临床试验中显示出治疗癌症的巨大希望。这种捕获和纯化将使用膜技术完成，这是Natrix separation Inc.核心业务的基础，有望最大限度地减少下游处理步骤的数量，并确保活性病毒的恢复。已知病毒的种类繁多及其独特的特性可能对开发强大的通用下游病毒制剂策略（特别是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。为了最大限度地减少不同的性质，本工作的重点是水疱性口炎病毒包膜糖蛋白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是VSV-G伪型的，以增加其效力。基于VSV-G的纯化策略，我们将使用Natrix技术平台对许多领先的治疗性候选病毒进行综合纯化。将同时审查两项主要战略。首先，使用Natrix，将制作一种肝素功能化的膜，用于捕获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。将评估这两种策略对病毒恢复、纯度和效力的影响。

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.