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治疗中的应用前景 多形性(GBM)。在目前的资助期间，我们设计了一系列新的HSV载体，能够安全但 通过(I)全载体重定向流行的人GBM受体(EGFR/EGFRvIII)有效地杀灭肿瘤 (Ii)细胞miR-124阻断正常脑内的载体复制(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)促进了动物模型中载体的扩散和肿瘤的分解。然而， 肿瘤中的载体复制仍然可以受到自然杀伤(NK)细胞的限制，自然杀伤细胞通常可以保护宿主免受HSV的感染 感染。项目2和项目4最近证明，干扰NK细胞的激活和杀伤 感染病毒的细胞通过允许更强大和更广泛的溶瘤病毒传播来增强GBM的治疗。我们 建议通过比较载体转基因介导的NK的影响来验证和扩展这些发现 阻断PPG的两个碱基载体KGN-4：T124(菌株KOS)和载体的溶瘤活性 RQNestin34.5(F株)，来自项目2，在三种不同的同基因小鼠GBM模型中。因为这些基地 载体具有不同的遗传背景，受不同的安全机制控制，它们的比较 这些实验对于选择最有效的骨干和武装未来的基因将是重要的 审判。我们将利用两个HSV允许的小鼠胶质瘤细胞系CT2A和GL261N4来产生脑瘤 在免疫功能正常的小鼠体内注射肿瘤细胞。此外，我们还与Eric建立了合作关系 Holland(华盛顿大学)验证他的遗传诱导的小鼠GBM模型的关键结果，该模型基于 RCAS/TV-A系统。在目标1中，我们将建立KGN-4：T124与KGN-4相比的基线肿瘤治疗效果 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组成的影响。在目标2中，我们将采取 利用这些基线数据来评估NK细胞拮抗在肿瘤治疗中的作用。在协作中 在项目2和项目4中，我们将确定携带编码可溶性形式的人巨细胞病毒基因的载体的效果。 UL141和/或PRV糖蛋白D可降低NK细胞识别的胶质瘤细胞配体的表达。在……里面 目的3，我们将表达另外两种巨细胞病毒衍生蛋白gp34和gp68的Fcγ结合胞外区。 已知能抑制NK细胞的激活。在与项目4的协作实验中，我们将检验假设 Sgp34将抑制经典的抗体依赖的细胞毒性(ADCC)，增强载体复制和 当肿瘤病毒裂解时，sgp34和/或sgp68会干扰Fc桥联细胞毒性(FcBCC)。我们还将 向项目3提供TIMP-3表达载体以抑制巨噬细胞间的Notch信号转导，从而 减少巨噬细胞对感染肿瘤细胞的识别，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.