Myxoma Virus (MV) Oncolysis for treating human cancer

项目摘要 粘液瘤病毒（MV）在体内仅感染兔，但也具有感染多种人类的天然能力。 癌细胞在体外和体内。因此，MV是用于治疗人类肿瘤的溶瘤病毒疗法的有吸引力的候选者。 癌MV已用于在异种移植中成功治疗几种不同的人类脑癌。 免疫缺陷小鼠和免疫活性小鼠中的鼠转移性黑素瘤。MV将在 通过利用一种治疗策略来开发用于人类临床试验的病毒， 适用于：体外清除人骨髓或患者的动员PBMC样品中的癌细胞 这些患者在高剂量化疗后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建议：1）MV不会干扰或 移植正常人的免疫缺陷小鼠中的人多能干细胞分化受损 2）MV清除了多种人类癌细胞， 体内净化，3）MV不仅可以有效地净化体内允许的白血病/淋巴瘤细胞， 出乎意料地阻止甚至非允许的人白血病细胞（如KG 1细胞）的植入，或 肿瘤诱导，和4）表达多种有用的报道蛋白（荧光和 生物发光的）用于成像目的。具体而言，我们的目标是： 1)体外治疗正常人造血干细胞的安全性： 将使用移植的免疫缺陷NOG测试正常人干细胞分化的MV清除 小鼠以验证完全造血细胞植入和免疫重建。MV吹扫将在 使用来自正常骨髓和G-CSF动员的PBMC的原代人干/祖细胞， 在体外造血集落形成细胞测定中以及在体内有效的血液细胞移植中， 2)优化MV离体癌细胞清除：两种人类癌症，B细胞淋巴瘤和急性髓系 将研究离体MV清除以消除其体内致瘤潜力的能力 在移植的NOG小鼠中。癌细胞和病毒将被标记上可区分的内切酶， 移植的肿瘤细胞和治疗性病毒将在体内独立追踪。原代细胞 急性髓性白血病患者也将接受MV特异性消除 污染癌细胞并允许仅非癌性人白细胞的选择性植入。 3)研究MV净化原代人白血病细胞的机制：我们最近发现， 用MV离体感染人KG 1白血病细胞阻止了随后的植入和肿瘤 尽管这些细胞是完全不允许的， 用于MV感染的体外试验。为了评估病毒诱导的细胞信号传导变化，我们探测了MV感染的KG 1细胞， 用一系列针对46种不同的人类信号磷蛋白的抗体，观察到MV感染 特异性诱导KG 1细胞中的Stat 5和Hck活化。我们将探讨这些功能的重要性 通过MV成功地体外清除人白血病细胞的宿主细胞信号传导激活。

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.