Myxoma Virus (MV) Oncolysis for treating human cancer

项目摘要 粘液瘤病毒(MV)不仅在体内感染兔，而且具有感染多种人的天然能力 体外和体内的癌细胞。因此，MV是一种有吸引力的治疗人类肿瘤的溶瘤病毒疗法。 癌症。MV已被成功地用于治疗几种不同类型的人类脑癌。 免疫缺陷小鼠和免疫活性小鼠的小鼠转移性黑色素瘤。在这里，MV将是 通过开发一种治疗策略来开发用于人类临床试验的病毒，这种治疗策略对病毒来说是独一无二的 适合：体外净化人骨髓中的癌细胞或从患者体内动员的PBMC样本 在大剂量化疗后，世卫组织通常被排除在自体干细胞移植之外。 已经收集了大量的初步数据来支持这一提议：1)MV不会扰乱或 移植正常人的免疫缺陷小鼠对人多能干细胞分化的影响 骨髓或细胞因子动员的PBMCs，2)MV消除多种人类癌细胞 体内清除，3)MV不仅可以有效清除体内允许的白血病/淋巴瘤细胞，而且还可以 出人意料地阻止甚至是不允许的人类白血病细胞(如KG1细胞)植入或 肿瘤诱导，以及4)表达多种有用报告蛋白(荧光和 生物发光)用于成像目的已经被构建。具体来说，我们的目标是： 1)验证MV对正常人造血干细胞体外治疗的安全性： 将使用植入的免疫缺陷NOG来测试MV净化对正常人类干细胞分化的作用 小鼠以验证完全的造血细胞植入和免疫重建。将在以下位置测试MV清除 来源于正常骨髓和G-CSF动员的PBMCs的原代人类干/祖细胞，使用 在体外进行造血祖细胞集落形成试验，以及在体内进行有效的血细胞植入。 2)优化MV体外癌细胞净化：B细胞淋巴瘤和急性髓系白血病两种人类癌症 白血病，将研究体外MV清除能力，以消除其体内的致瘤潜力 植入NOG小鼠体内。癌细胞和病毒将被标记上可区分的荧光素酶，从而 植入的肿瘤细胞和治疗性病毒将在体内独立追踪。原代细胞来自 急性髓系白血病患者还将接受MV的能力测试，以特异性消除 污染癌细胞，只允许有选择地植入非癌症的人类白细胞。 3)研究MV对原代人类白血病细胞的净化机制：我们最近发现 MV体外感染人KG1白血病细胞可防止随后的植入和肿瘤 将这些细胞形成NOG受体小鼠，尽管这些细胞是完全不允许的 在体外对MV的感染。为了评估病毒诱导的细胞信号变化，我们探测了感染MV的KG1细胞 用一组针对46种不同人类信号磷酸蛋白的抗体，观察到MV感染 在KG1细胞中特异性地诱导Stat5和HCK的激活。我们将探讨这些功能的意义 MV激活宿主细胞信号为肿瘤细胞成功清除人类白血病提供依据。

Project Summary Myxoma virus (MV) infects only rabbits in vivo, but also has a natural capacity to infect a wide variety of human cancer cells in vitro and in vivo. Thus, MV is an attractive candidate for oncolytic virotherapy to treat human cancer. MV has been used to successfully treat several diverse human brain cancers in xenografted immunodeficient mice and murine metastatic melanoma in immunocompetent mice. Here, MV will be developed for human clinical trials by exploiting a therapeutic strategy for which the virus is uniquely well- suited: ex vivo purging of cancer cells within human bone marrow or mobilized-PBMC samples from patients who would normally be excluded from autologous stem cell transplantation following high dose chemotherapy. Considerable preliminary data has been collected to support this proposal: 1) MV does not perturb or compromise human multipotent stem cell differentiation in immunodeficient mice engrafted with normal human bone marrow or cytokine-mobilized PBMCs, 2) MV eliminates a wide variety of human cancer cells following ex vivo purging, 3) MV can effectively purge not only permissive leukemia/lymphoma cells in vivo, but also unexpectedly prevents even nonpermissive human leukemia cells (such as KG1 cells) from engraftment or tumor induction, and 4) MV recombinants that express a variety of useful reporter proteins (fluorescent and bioluminescent) for imaging purposes have already been constructed. Specifically, our aims are: 1) Validate MV safety for ex vivo treatment of normal human hematopoietic stem cells: The safety of MV-purging for normal human stem cell differentiation will be tested, using engrafted immunodeficient NOG mice to verify full hematopoietic cell engraftment and immune reconstitution. MV purging will be tested on primary human stem/progenitor cells derived from normal bone marrow and G-CSF-mobilized PBMCs, using hematopoietic colony forming cell assays in vitro as well as for efficient hematologic cell engraftment in vivo. 2) Optimize MV ex vivo cancer cell purging: Two human cancers, B-cell lymphoma and acute myeloid leukemia, will be investigated for the ability of ex vivo MV purging to eliminate their tumorigenic potential in vivo in engrafted NOG mice. The cancer cells and viruses will be tagged with distinguishable luciferases that allow the engrafted tumor cells and the therapeutic virus to be independently tracked in vivo. Primary cells from acute myeloid leukemia patients will also be tested for the ability of MV to specifically eliminate the contaminating cancer cells and allow the selective engraftment of only noncancerous human leukocytes. 3) Investigate the mechanism of MV purging of primary human leukemia cells: We have recently shown that ex vivo infection of human KG1 leukemia cells with MV prevents the subsequent engraftment and tumor formation of these cells into NOG recipient mice, despite the fact that these cells are completely nonpermissive for MV infection in vitro. To assess for virus-induced cell signaling changes, we probed MV-infected KG1 cells with an array of antibodies to 46 different human signaling phosphoproteins, and observed that MV infection specifically induces Stat5 and Hck activation in KG1 cells. We will explore the functional significance of these host cell signaling activations for the successful ex vivo tumor cell purging of human leukemia calls by MV.