Developing diagnostic and therapeutic stem cells for cancer therapy

描述（由申请人提供）：最近的证据表明胚胎和成体干细胞在细胞治疗中具有巨大的治疗潜力。在先前的研究中，我们已经确定：a)治疗工程干细胞广泛迁移到肿瘤和浸润性脑沉积物中，并在移植到胶质瘤小鼠模型中时具有凋亡和抗血管生成作用；b)受体靶向抗肿瘤治疗的动态和干细胞的命运可以在体内实时可视化。在这个提议中，我们将创造出抗毒素的人间充质干细胞（MSC），用于靶向纳米体和细胞毒性药物的现场递送，同时阻止肿瘤细胞的增殖和诱导杀伤，而不影响正常的大脑。具体来说，MSC将被设计成表达靶向治疗蛋白，针对过表达的EGFR，并在胶质瘤细胞中特异性表达IL13Ra2和死亡受体（DR）4/5。我们与Henegouwen实验室（荷兰乌得勒支大学）密切合作，该实验室在开发低分子量、高可溶性EGFR特异性纳米体（EGFR- nb）方面处于领先地位，我们最近表明，哺乳动物细胞可以用来表达可分泌的二价EGFR纳米体。我们将首先在MSC中表达EGFR- nb，并在一组原发性胶质瘤细胞和CD133+原发性脑肿瘤细胞中研究EGFR- nb对EGFR信号传导和细胞增殖的影响，目前正在与settman实验室（波士顿MGH）合作。许多研究表明，当EGFR信号拮抗剂与细胞因子联合使用时，具有协同抗肿瘤作用。将测试两种基于选择性靶向胶质瘤细胞的不同细胞毒性疗法。在第一种方法中，我们将与Rich实验室（B&W医院，波士顿）合作创建抗毒素MSC，并设计表达EGFR-NB和il13r靶向白喉毒素（DT）的MSC，已知其通过延伸因子-2 （EF-2）的adp核糖基化抑制蛋白质合成而诱导细胞死亡。在第二种方法中，我们将创建表达EGFR-NB和S-TRAIL的MSC，我们已经对其进行了广泛的表征，并证明它们可以通过增殖胶质瘤细胞中上调的死亡受体(DR)-4/5选择性地诱导细胞凋亡。这两种方法都将在已建立的胶质瘤系的培养和体内测试其有效性。基于这些发现，我们将在高度侵袭性胶质瘤原发小鼠模型中使用最有效的MSC治疗。我们假设，治疗性间充质干细胞的现场递送将导致细胞存活途径的同时下调和死亡途径的激活，从而增强胶质瘤的根除。与Weissleder实验室（波士顿MGH）密切合作，整合基因工程荧光和生物发光成像标记以及体内成像将使我们能够跟踪MSC的递送和命运，并评估其体内治疗效果。这些研究预计将对开发新型干细胞疗法产生重大影响，最终将与临床试验相兼容。

DESCRIPTION (provided by applicant): Recent evidence indicates that both embryonic and adult stem cells have enormous therapeutic potential for cell therapy. In prior research, we have established that: a) therapeutically engineered stem cells migrate extensively to tumors and to infiltrating deposits in the brain and have apoptotic and antiangiogenic effects when transplanted into mouse models of glioma; and b) the dynamics of receptor targeted anti-tumor therapies and fate of stem cells can be visualized in real time in vivo. In this proposal, we will create toxin resistant human mesenchymal stem cells (MSC) for on-site delivery of targeted nanobodies and cytotoxic agents to simultaneously block proliferation and induce killing of tumor cells without affecting the normal brain. Specifically, MSC will be engineered to express targeted therapeutic proteins directed against overexpressed EGFR and specifically expressed IL13Ra2 and death receptors (DR)4/5 in glioma cells. In close collaboration with Henegouwen lab (Utrecht University, The Netherlands) which is leading efforts in developing low molecular weight, highly soluble EGFR specific nanobodies (EGFR-NB), we have recently shown that mammalian cells can be employed to express secretable bivalent EGFR nanobodies. We will initially express EGFR-NB in MSC and study the effect of EGFR-NB on EGFR signaling and cell proliferation in a panel of primary glioma cells and CD133+ primary brain tumor cells in an ongoing collaboration with Settleman lab (MGH, Boston). A number of studies have shown synergistic anti-tumor effects when EGFR signaling antagonists are combined with cytokines. Two different cytotoxic therapies based on selectively targeting glioma cells will be tested. In the first approach we will create toxin resistant MSC in an ongoing collaboration with Rich lab (B&W Hospital, Boston) and engineer MSC expressing EGFR-NB and IL13R-targeted Diphtheria toxin (DT) which is known to induce cell death by inhibition of protein synthesis through ADP-ribosylation of elongation factor-2 (EF-2). In the second approach we will create MSC expressing EGFR-NB and S-TRAIL, which we have extensively characterized and shown to selectively induce apoptosis via up-regulated death receptors (DR)-4/5 in proliferating glioma cells. Both approaches will be tested for their efficacy in culture and in vivo in established glioma lines. Based on these findings, we will utilize the most efficient therapeutic MSC in a highly invasive primary mouse model of glioma. We hypothesize that on site delivery of therapeutic MSC will result in simultaneous down-regulation of cell survival pathways and activation of death pathways thus resulting in enhanced eradication of gliomas. The integration of genetically engineered fluorescent and bioluminescent imaging markers and in vivo imaging in close collaboration with Weissleder lab (MGH, Boston) will allow us to follow delivery and fate of MSC and to asses their therapeutic efficacy in vivo These studies are expected to have a major impact in developing novel stem cell therapies that will eventually be compatible with clinical trials.