Reagents for Targeted Ablation of Residual Contaminating Pluripotent Stem Cells

用于残留污染多能干细胞靶向消融的试剂

• 批准号: 8786795
• 负责人: Dana Larocca
• 金额: $ 27.03万
• 依托单位: BIOTIME, INC.
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8786795
• 关键词: Reagents; Targeted; Ablation; Residual; Contaminating

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.

描述（由申请人提供）：人类多能茎（HPS）细胞具有巨大的潜力，作为可再生细胞的可再生细胞来源，用于发现药物发现，疾病建模和移植以取代失去退化性疾病或损伤的细胞。对 HPS细胞研究的成功应用是缺乏产生HPS细胞衍生物纯种群的方法。污染细胞既是药物发现的问题，在该药物发现​​中，需要纯粹的种群以进行测定可重复性，也需要移植，因为它们可能存在肿瘤形成或与不适当的细胞类型的分化，它们会出现安全问题。具体而言，我们在这里建议解决具有形成畸胎瘤潜力的残留污染未分化的HPS细胞的问题。我们建议开发靶向毒素的HPS细胞（HPS-CTT），这些毒素杀死残留的HPS细胞，其损伤最小或分化细胞群的改变。为了达到选择性细胞靶向毒性，W将结合内在化的HPS选择性靶向剂与植物毒素，saporin，这是 仅在细胞内在化时有毒。这种方法比以前提出的遗传修饰和物理分离方法要简单得多，物理分离方法需要细胞操作，以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，因为任何内在化抗体或肽都可以适应靶向细胞杀死。此外，HPS-CTT设计为用死细胞去除，从而降低了其对存活细胞的潜在影响。为了证明I阶段的可行性，我们将筛选抗体和肽的选择性HPS细胞靶向。细胞靶向剂（CTA）将间接连接至毒素，并测试了它们在模型细胞系统中除去未分化的HS细胞的能力，该模型细胞系统中含有分化的HPS细胞的混合物，该细胞与未分化的HPS细胞飙升。我们将通过流式细胞仪，最多的和定量的RT-PCR测量HPS细胞含量。最佳HPS-CTT去除HPS细胞的能力将使用体内畸胎瘤测定法测试。 HPS-CTT在治疗人群中的持久性将通过Western印迹分析和功能测定法测量，以检测残留毒性。将各种靶向剂与次级毒素偶联物相结合的2个组件系统将在I阶段使用以显示可行性。在第二阶段，我们将开发直接偶联到毒素的细胞靶向剂，并进一步优化HPS细胞去除方案。