Mengovirus Replicon for Enhanced Oncolytic Virotherapy

摘要 溶瘤病毒疗法作为一种癌症治疗的潜力已经在临床试验中得到了清楚的证明， 然而，目前可用的溶瘤病毒的总体效果不是很好。很明显，一个 这种治疗方式的溶瘤期和免疫期之间的平衡需要在 以最大限度地发挥治愈的潜力。孟戈病毒，一种微小核糖核酸病毒病原体，代表了一类改进的 溶瘤病毒，因为它能够在具有免疫能力的动物身上进行临床前评估，包括 老鼠和灵长类动物。孟加拉病毒可以引起脑炎和心肌炎，然而，我们通过以下方法增强了其安全性 截断多胞苷束(一种致病决定因素)，并结合神经元和心脏特异性 病毒基因组中的microRNA。用这种双重去靶向病毒vMC24-NC治疗后， 同基因小鼠多发性骨髓瘤浆细胞瘤的消退。然而，转移复发是 在接受治疗的小鼠中观察到了50%，这表明需要改善这种病毒疗法的免疫阶段。 这项提议的目标是开发一种表达免疫刺激蛋白的巨细胞病毒疗法 在溶瘤阶段增强免疫期。外源基因导入小核糖核酸病毒的研究 基因组因其有限的插入能力而受到抑制。因此，我们开发了一个基于孟戈病毒的复制子 其中编码衣壳蛋白的基因被分泌的荧光素酶基因取代。此复制图标 导致外源基因表达、基因组复制和封装，当联合 使用vMC24-NC进行管理。我们假设我们可以改善复制子结构的总体分布 通过创建具有编码衣壳蛋白和/或 根据复制子和辅助/病毒的剂量、比例和顺序优化治疗方案 行政管理。这些研究将首次产生一种有效和安全的单发系统 溶瘤小核糖核酸病毒可以携带免疫刺激蛋白并在具有免疫能力的动物身上进行测试 从而产生了优越的溶瘤病毒疗法。有鉴于此，我们提出了以下具体目标： 目的1.比较我们的衣壳替代(载体)复制子的传播效率和遗传稳定性 与a)复制能力强的vMC24-NC或b)编码vMC24-NC衍生衣壳的细胞共感染(辅助) 与载体复制子共同依赖于非结构蛋白功能的复制子。 目的2.确定肿瘤内载体基因组表达和传播的效率如何受以下因素的影响 载体和辅助体的相对剂量(II)载体和辅助体的时间序列。 目的3.评价免疫刺激性巨细胞病毒联合治疗的疗效 带有vMC24-NC或辅助复制体的复制体。

ABSTRACT The potential of oncolytic virotherapy as a cancer treatment has been clearly demonstrated in clinical trials, however the overall efficacies of currently available oncolytic viruses is modest. It has become clear that a balance between the oncolytic phase and immune phase of this treatment modality needs to be reached in order to maximize the potential of cure. Mengovirus, a picornaviral pathogen, represents an improved class of oncolytic viruses because of its ability to undergo preclinical evaluation in immunocompetent animals including mice and primates. Mengovirus can cause encephalitis and myocarditis, however, we enhanced its safety by truncating the polycytidine tract (a pathogenic determinant) and by incorporating neuron and cardiac specific microRNAs into the viral genome. Treatment with this dual-detargeted virus, vMC24-NC, results in complete regression of syngeneic mouse multiple myeloma plasmacytomas. However, metastatic recurrence was observed in 50% of the treated mice indicating a need for improving the immune phase of this virotherapy. The goal of this proposal is to develop a Mengovirus therapy expressing immunostimulatory proteins in order to boost the immune phase during the oncolytic phase. Incorporation of foreign genes into picornaviral genomes is inhibited by their limited insertion capacity. Therefore we developed a Mengovirus-based replicon where the genes encoding the capsid proteins were replaced with a secreted luciferase gene. This replicon results in foreign gene expression, genome replication, and encapsidation with short-term spread when co- administered with vMC24-NC. We hypothesize that we can improve the overall spread of the replicon construct by creating a co-dependence system with an additional helper replicon encoding the capsid proteins and/or optimizing the treatment protocol based on dose, ratio, and sequence of replicon and helper/virus administration. These studies will produce for the first time a single-shot system where a potent and safe oncolytic picornavirus can be armed with immunostimulatory proteins and tested in immunocompetent animals resulting in a superior oncolytic virotherapy. With this in mind, we propose the following specific aims: Aim 1. Compare the efficiency of spread and genetic stability of our capsid-substituted (vector) replicons in cells coinfected with a) replication-competent vMC24-NC or b) capsid-encoding vMC24-NC derived (helper) replicons that are co-dependent with the vector replicon for nonstructural protein functions. Aim 2. Determine how the efficiency of intratumoral vector genome expression and spread is impacted by (i) relative dose of vector and helper (ii) temporal sequencing of vector and helper. Aim 3. Evaluate the therapeutic efficacy of combination therapies using immunostimulatory Mengovirus replicons with vMC24-NC or helper replicons.