Enhancing Delivery and Regulating Gene Expression in Oncolytic Vaccinia Vectors

描述(申请人提供)：我们之前设计并实现了基于痘苗病毒的多种溶瘤载体，开发了一组基因缺失和肿瘤靶向的菌株，显示了良好的临床前和早期临床结果。我们还率先使用基于细胞的载体来更有效地将溶瘤病毒株输送到他们的肿瘤靶点。特别是，我们证明了细胞因子诱导的杀伤(CIK)细胞不仅可以作为痘苗病毒株的有效载体，而且还可以与病毒协同作用以增强其抗肿瘤效果。尽管最近取得了这些进展，但这些疗法仍然没有充分发挥其潜力。在这项提案中，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才能有效地重复使用相同的疗法；以及需要在这些载体中同时增加效力和改善安全性。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，我们将专注于改善病毒载体对肿瘤的系统输送，特别是在预先免疫的动物模型中，并寻求实施甚至利用抗病毒免疫反应的策略；在最终目标中，我们将检查将不同的转基因或基本病毒基因产品置于外部调节的蛋白质稳定结构域控制之下的优势。在面对抗病毒免疫反应时，总共将采用三种免费的方法来加强病毒的传递，重点是避开循环中和抗体；(I)表位交换或表位移除将用于减少抗体识别，(Ii)病毒颗粒与目标微泡结合，随后在肿瘤环境中超声介导释放，以及(Iii)使用细胞载体将病毒输送到肿瘤，特别是加入具有突变的病毒株，这会增加原位产生的病毒的“隐形”或包膜形式的水平。在最终目标中，我们将进一步定义和整合先前证明的外部调节从病毒载体表达的不同治疗性转基因的功能的优点。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，这项提议的总体目标是克服溶瘤病毒疗法的一些主要局限性，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选择，包括复发、耐药和播散性疾病。该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，因此我们预计它将直接为患者带来好处。 与公共卫生相关：这项提案的总体目标是加强溶瘤(复制选择性)病毒的应用。这将主要涉及改善这些载体在NAOVE和以前免疫的宿主中的系统递送。此外，我们将整合受调控的转基因功能，以提高这些疗法的有效性，我们还将寻求将溶瘤病毒与免疫细胞疗法相结合。这项提议的重点不仅是将生物制剂作为单一疗法加以改进，还将重点放在如何将它们结合起来，以便利用病毒和免疫细胞疗法之间的自然相互作用来提高治疗结果。

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.