Enhancing Delivery and Regulating Gene Expression in Oncolytic Vaccinia Vectors

描述（由申请人提供）：我们之前设计并实施了多种基于牛痘病毒的溶瘤载体，开发了一组基因缺失和肿瘤靶向菌株，这些菌株在临床前和早期临床结果中显示出有希望。我们还率先使用基于细胞的载体载体，更有效地将溶瘤病毒株运送到其肿瘤目标。特别是，我们证明了细胞因子诱导的杀伤细胞（CIK）细胞不仅可以作为牛痘菌株的有效递送载体，而且可以与病毒协同作用以增强其抗肿瘤作用。尽管最近取得了这些进展，但这些疗法仍未充分发挥其潜力。在本建议中，我们确定了这些生物疗法未来发展的关键限制，包括需要克服抗病毒免疫反应，以便有效地重复使用相同的疗法；需要在这些载体中增加效力和提高安全性。我们希望在三个主要目标中解决这些问题：在前两个目标中，我们将重点关注改善病毒载体对肿瘤的全身递送，特别是在预免疫动物模型中，并寻求实施策略，甚至利用抗病毒免疫反应；在最后的目的中，我们将研究将不同的转基因或必要的病毒基因产物置于外部调节的蛋白质稳定域控制下的优势。在面对抗病毒免疫反应时，将采用三种互补方法来增强病毒传递，重点是逃避循环中和抗体；(i)表位交换或表位移除将用于降低抗体识别，（ii）将病毒颗粒与靶向微泡结合，随后在肿瘤环境中进行超声介导释放，以及（iii）使用细胞载体将病毒传递到肿瘤，特别是结合具有突变的病毒株，这些突变增加了原位产生的“隐形”或包膜形式的病毒水平。在最后的目标中，我们将进一步定义和整合先前证明的从病毒载体表达的不同治疗性转基因的外部调节功能的优势。或者，我们将调节必要的病毒基因表达，从而在载体内提供一个关闭开关。因此，本提案的总体目标是克服溶瘤病毒治疗的一些主要局限性，从而提高其临床潜力，并为一些最难治疗的癌症患者提供现实的替代治疗方案，包括复发、耐药和播散性疾病。本提案中产生的数据将直接应用于匹兹堡大学正在开发的临床试验的方案设计，因此我们希望它能直接给患者带来好处。

DESCRIPTION (provided by applicant): We have previously designed and implemented a variety of oncolytic vectors based on vaccinia virus, developing a panel of gene-deleted and tumor-targeting strains that have displayed promising pre- clinical and early clinical results. We have also pioneered the use of cell-based carrier vehicles to more efficiently deliver oncolytic virus strains to their tumor targets. In particular we demonstrated that Cytokine Induced Killer (CIK) cells could not only act as effective delivery vehicles for vaccinia strains, but could also synergize with the virus to increase their anti-tumor effects. Despite these recent advances, these therapies have still not reached their full potential. In this proposal we have identified key limitations to the future development of these biological therapies, including the requirement to overcome anti-viral immune responses in order to efficiently repeat treat with the same therapy; and the need for both added potency and improved safety within these vectors. We look to address these in three main aims: in the first two aims we will focus on improving the systemic delivery of the viral vector to the tumor, especially in pre-immunized animal models and look to implement strategies to even take advantage of the anti-viral immune response; in the final aim we will examine the advantages of placing different transgene or essential viral gene products under the control of externally regulated protein stability domains. A total of three complimentary approaches will be applied to enhance viral delivery in the face of an anti-viral immune response, focusing on evading circulating neutralizing antibody; (i) epitope swapping or epitope removal will be used to reduce antibody recognition, (ii) binding of viral particles to targeted microbubbles, followed by ultrasound mediated release within the tumor environment and (iii) use of cell carriers to deliver virus to the tumor, particularly incorporating viral strains with mutations that increase the levels of the 'stealth' or enveloped form of the virus produced in situ. In the final aim we will further define and incorporate previously demonstrated advantages of externally regulating the function of different therapeutic transgenes expressed from the viral vectors. Alternatively, we will regulate essential viral gene expression, so providing a shut-off switch within the vector. The overall objectives of this proposal are therefore to overcome some of the major limitations of oncolytic viral therapy, so improving its clinical potential and providing realistic alternative treatment options for patients with some of the hardest to treat cancers, including relapsed, drug-resistant and disseminated disease. Data produced in this proposal will be directly applied to the protocol design of clinical trials under development at the University of Pittsburgh, and so we expect it to directly lead to patient benefits. PUBLIC HEALTH RELEVANCE: This overall aim of this proposal is to enhance the application of oncolytic (replication selective) viruses. This will primarily involve improving the systemic delivery of these vectors, both in naove and previously immunized hosts. In addition we will incorporate regulated transgene function to improve the effectiveness of these therapies and we will look to integrate the oncolytic viruses with immune cell therapies. The focus of this proposal will not only be on improving the biological agents as single therapies, but also on how to combine them such that the natural interactions between viruses and immune cell therapies can be utilized to enhance the therapeutic outcome.