Developing diagnostic and therapeutic stem cells for cancer therapy

描述(由申请人提供)：最近的证据表明，胚胎和成人干细胞在细胞治疗方面都具有巨大的治疗潜力。在先前的研究中，我们已经确定：a)治疗工程干细胞在移植到小鼠脑胶质瘤模型中时，可以广泛地迁移到肿瘤和脑内的浸润性沉积物中，并具有凋亡和抗血管生成的作用；b)可以在体内实时观察受体靶向抗肿瘤治疗的动态和干细胞的命运。在这项提议中，我们将创造具有毒素抗性的人间充质干细胞(MSC)，用于现场输送靶向纳米体和细胞毒剂，以同时阻止增殖和诱导杀伤肿瘤细胞，而不影响正常大脑。具体地说，MSC将被设计为表达针对过度表达的EGFR的靶向治疗蛋白，并在胶质瘤细胞中特异性表达IL13RA2和死亡受体(DR)4/5。在与荷兰乌得勒支大学Henegouwen实验室的密切合作下，我们最近证明，哺乳动物细胞可以表达可分泌的二价EGFR纳米体。Henegouwen实验室是开发低分子、高可溶性EGFR特异性纳米体的领先者。我们将首先在MSC中表达EGFR-NB，并与Settleman实验室(MGH，波士顿)持续合作，研究EGFR-NB对一组原代胶质瘤细胞和CD133+原代脑瘤细胞中EGFR信号和细胞增殖的影响。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疗法。在第一种方法中，我们将与Rich Lab(波士顿B&W医院)持续合作创建耐毒素的MSC，并设计表达EGFR-NB和IL13R靶向白喉毒素(DT)的MSC，已知DT通过延长因子-2(EF-2)的ADP-核糖化抑制蛋白质合成而诱导细胞死亡。在第二种方法中，我们将建立表达EGFR-NB和S-TRAIL的MSC，我们已经广泛地表征并表明，MSC通过上调死亡受体(DR)-4/5在增殖的胶质瘤细胞中选择性地诱导凋亡。这两种方法都将在培养和体内对已建立的胶质瘤系进行有效性测试。基于这些发现，我们将利用最有效的治疗MSC来治疗高侵袭性的原代小鼠脑胶质瘤模型。我们假设，就地输送治疗性MSC将导致细胞生存途径的下调和死亡途径的激活，从而促进胶质瘤的根除。与Weissleder实验室(波士顿MGH)密切合作，将基因工程荧光和生物发光成像标记与体内成像相结合，将使我们能够跟踪MSC的交付和命运，并在体内评估它们的治疗效果。这些研究有望对开发最终与临床试验兼容的新型干细胞疗法产生重大影响。 公共卫生相关性：在这项建议中，我们将设计人间充质干细胞(MSC)来表达针对胶质瘤细胞中过度表达的受体的靶向治疗蛋白。将探索两种不同的联合治疗方法，基于靶向胶质瘤细胞的细胞增殖和死亡途径。所开发的药物和策略将被设计为可在临床上翻译，并将对开发有效的脑瘤治疗方法产生重大影响。

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. PUBLIC HEALTH RELEVANCE: In this proposal we will engineer human mesenchymal stem cells (MSC) to express targeted therapeutic proteins directed against overexpressed receptors in glioma cells. Two different combinatorial therapy approaches based on targeting cell proliferation and death pathways in glioma cells will be explored. The developed agents and strategies will be designed to be clinically translatable and should have a major impact in developing efficient therapies for brain tumors.