Reagents for Targeted Ablation of Residual Contaminating Pluripotent Stem Cells

描述(申请人提供)：人类多能干细胞(HPS)在药物发现、疾病建模和移植以取代因退行性疾病或损伤而失去的细胞方面具有巨大的可再生细胞来源的潜力。一个关键的障碍是 HPS细胞研究的成功应用在于缺乏生产HPS细胞衍生物的纯群体的方法。污染细胞既是药物发现的问题，也是移植的一个问题，在药物发现中，需要纯净的种群来实现检测的重复性，而在移植中，由于它们有可能形成肿瘤或分化为不适当的细胞类型，因此存在安全问题。具体地说，我们建议在这里解决残留污染有可能形成畸胎瘤的未分化HPS细胞的问题。我们建议开发HPS细胞靶向毒素(HPS-CTTS)，以最小的损害或改变分化的细胞群来杀死残留的HPS细胞。为了实现选择性细胞靶向毒性，W将内化HPS选择性靶向剂与植物毒素Saporin结合，Saporin是 只有在细胞内化时才是有毒的。这种方法比以前提出的遗传修饰和物理分离方法简单得多，这些方法需要细胞操作，可能会改变或强调所需的细胞群体。它也比以前提出的细胞毒性抗体更灵活，因为任何内化的抗体或多肽都可以适应靶向细胞杀伤。此外，HPS-CTT被设计为与死亡细胞一起移除，从而减少其对存活细胞的潜在影响。为了证明在第一阶段的可行性，我们将筛选抗体和多肽用于选择性HPS细胞靶向。细胞靶向剂(CTA)将间接连接到毒素上，并在包含已分化的HPS细胞与未分化的HPS细胞的混合物的模型细胞系统中测试它们去除未分化的HS细胞的能力。采用流式细胞仪、PluriTest和定量RT-PCR检测治疗前后HPS细胞含量。最佳HPS-CTT去除HPS细胞的能力将通过体内畸胎瘤试验进行测试。HPS-CTT在治疗人群中的持久性将通过蛋白质印迹分析和检测残留毒性的功能试验来衡量。在第一阶段将使用一个结合了各种靶向剂和次级毒素结合物的两组分系统来显示可行性。在第二阶段，我们将开发与毒素直接偶联的细胞靶向药物，并进一步优化HPS细胞去除方案。

DESCRIPTION (provided by applicant): Human pluripotent stem (hPS) cells have great potential as a renewable source of cells for drug discovery, disease modeling and for transplantation to replace cells lost to degenerative diseases or injury. A critical barrier to the successful application of hPS cell research is the lack of methods for producing pure populations of hPS cell derivatives. Contaminating cells are both an issue for drug discovery where pure populations are needed for assay reproducibility and for transplantation where they present a safety issue because of their potential for tumor formation or differentiation to inappropriate cell types. Specifically, we propose here to address the problem of residual contaminating undifferentiated hPS cells that have the potential to form teratomas. We propose to develop hPS cell targeted toxins (hPS-CTTs) that kill residual hPS cells with minimal damage or alteration of the differentiated cell population. To achieve selective cell targeted toxicity, w will combine an internalizing hPS selective targeting agent with the plant toxin, saporin, which is only toxic upon cellular internalization. This approach is much simpler than previously proposed genetic modification and physical separation methods that require cell manipulations that could alter or stress the desired cell population. It is also much more flexible than the previously proposed cytotoxic antibody because any internalizing antibody or peptide can be adapted for targeted cell killing. Moreover, the hPS-CTT is designed to be removed with the dead cells thus reducing its potential impact on the surviving cells. To demonstrate feasibility in phase I, we wil screen antibodies and peptides for selective hPS cell targeting. The cell targeting agents (CTAs) will be indirectly conjugated to a toxin, and tested for their ability to remove undifferentiated hS cells in a model cell system containing admixtures of differentiated hPS cells spiked with undifferentiated hPS cells. We will measure hPS cell content before and after treatment by flow cytometry, the PluriTest, and quantitative RT-PCR. The ability to the optimal hPS-CTT to remove hPS cells will be tested using an in vivo teratoma assay. The persistence of the hPS-CTT in the treated population will be measured by western blot analysis and a functional assay to detect residual toxicity. A 2 component system that combines various targeting agents with secondary toxin conjugates will be used in phase I to show feasibility. In phase II, we will develop cell targeting agents directly conjugated to the toxin and further optimize protocols for hPS cell removal.