Project 1: Arming Oncolytic HSV Vectors to Improve Virolysis in Syngeneic Mouse Models of GBM

项目摘要-项目1 溶瘤单纯疱疹病毒（oHSV）载体在胶质母细胞瘤的治疗中提供了相当大的希望 多形性（GBM）。在目前的资助期内，我们设计了一系列新的HSV载体，能够安全， 通过（i）全载体重靶向普遍存在的人GBM受体（EGFR/EGFRvIII）的有效肿瘤破坏 （ii）正常脑中载体复制的细胞miR-124阻断（KGN-4：T124：我们的基础载体）， 或（iii）这些特征的组合（KGNE-4：T124）。用矩阵进一步武装组合向量 金属蛋白酶（KGNE-4：T124-MMP9）在动物模型中增强载体扩散和溶瘤。然而，在这方面， 肿瘤中的载体复制仍然受到自然杀伤（NK）细胞的限制，自然杀伤细胞通常保护宿主免受HSV的侵害 感染项目2和4最近表明，干扰NK细胞活化和杀伤NK细胞是一种有效的方法。 病毒感染的细胞通过允许更强和更广泛的溶瘤病毒扩散来增强GBM治疗。我们 我建议通过比较载体转基因介导的NK细胞的影响来验证和扩展这些发现， 阻断该PPG的两种基本载体，我们的KGN-4：T124（菌株科斯）和载体的溶瘤活性 来自项目2的rQNestin34.5（菌株F），在三种不同的GBM同基因小鼠模型中。因为这些基地 病媒具有不同的遗传背景，受不同的安全机制控制， 在这些实验中，选择最有效的骨干和武装基因的未来将是重要的 审判我们将利用两种HSV-容许的小鼠胶质瘤细胞系CT2A和GL261N4来产生脑肿瘤 在免疫活性小鼠中通过肿瘤细胞注射。此外，我们与Eric建立了合作关系， 荷兰（U.（华盛顿），以验证他的遗传诱导的GBM小鼠模型的关键结果， RCAS/tv-a系统。在目标1中，我们将确定KGN-4的基线肿瘤治疗疗效：T124与 rQNestin34.5.我们将进行肿瘤治疗剂量反应分析，并表征和比较 两种载体对肿瘤微环境（TME）组成的影响。在目标2中，我们将 这些基线数据的优点是评估NK细胞拮抗作用对肿瘤治疗的影响。合作 在项目2和4中，我们将确定携带编码可溶性HCMV的基因的载体的效果 UL141和/或PRV糖蛋白D，其降低NK细胞识别的神经胶质瘤细胞配体的表达。在 目的3，我们将表达另外两种HCMV衍生蛋白gp34和gp68的Fc γ结合胞外域， 已知抑制NK细胞活化。在与项目4的合作实验中，我们将测试假设 sgp34将抑制经典ADCC（抗体依赖性细胞毒性），增强载体复制， 肿瘤病毒裂解，而sgp34和/或sgp68将干扰Fc桥接细胞毒性（FcBCC）。我们还将 向项目3提供TIMP-3表达载体，以抑制巨噬细胞中的Notch信号传导， 减少巨噬细胞对受感染肿瘤细胞的识别并延长oHSV的持续时间。

PROJECT SUMMARY – PROJECT 1 Oncolytic herpes simplex virus (oHSV) vectors offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In the current funding period we designed a new series of HSV vectors capable of safe but efficient tumor destruction through (i) full vector retargeting to prevalent human GBM receptors (EGFR/EGFRvIII) (vector KGNE), (ii) cellular miR-124 blockade of vector replication in normal brain (KGN-4:T124: our base vector), or (iii) a combination of these features (KGNE-4:T124). Further arming of the combination vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in animal models. However, vector replication in tumors can still be limited by Natural Killer (NK) cells that normally protect the host from HSV infection. Projects 2 and 4 have recently demonstrated that interference with NK cell activation and killing of virus-infected cells enhances GBM treatment by allowing more robust and broader oncolytic virus spread. We propose to validate and extend these findings by comparing the impact of vector transgene-mediated NK blockade on the oncolytic activities of the two base vectors of this PPG, our KGN-4:T124 (strain KOS) and vector rQNestin34.5 (strain F) from Project 2, in three distinct syngeneic mouse models of GBM. Because these base vectors have different genetic backgrounds and are controlled by different safety mechanisms, their comparison in these experiments will be important for choosing the most effective backbone and arming genes for future trials. We will exploit two HSV-permissive mouse glioma cell lines, CT2A and GL261N4, to generate brain tumors in immunocompetent mice by tumor cell injection. In addition, we have an established collaboration with Eric Holland (U. of Washington) to verify key results in his genetically induced mouse models of GBM based on the RCAS/tv-a system. In Aim 1, we will establish base-line tumor treatment efficacies for KGN-4:T124 versus rQNestin34.5. We will perform tumor-treatment dose-response analyses, and characterize and compare the effects of the two vectors on the composition of the tumor microenvironment (TME). In Aim 2, we will take advantage of these base-line data to evaluate the effect of NK cell antagonism on tumor therapy. In collaboration with Projects 2 and 4, we will determine the effects of vector arming with genes encoding soluble forms of HCMV UL141 and/or PRV glycoprotein D that reduce the expression of glioma cell ligands recognized by NK cells. In Aim 3, we will express the Fcγ-binding ectodomains of two other HCMV-derived proteins, gp34 and gp68, that are known to inhibit NK cell activation. In collaborative experiments with Project 4, we will test the hypothesis that sgp34 will inhibit classical ADCC (antibody-dependent cellular cytotoxicity), enhance vector replication and tumor virolysis while sgp34 and/or sgp68 will interfere with Fc bridging cellular cytotoxicity (FcBCC). We will also provide TIMP-3 expression vectors to Project 3 to inhibit Notch signaling among macrophages and thereby reduce macrophage recognition of infected tumor cells and prolong oHSV persistence.