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

基本信息

批准号：
8455044
负责人：
Dana Larocca
金额：
$ 1.25万
依托单位：
MANDALA BIOSCIENCES, LLC
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-01 至 2013-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8455044
关键词：
Ablation Address Admixture Aftercare Alzheimer&apos s Disease Antibodies Binding Biological Assay Cell Death Cell Extracts Cell Line Cell Separation Cell Therapy Cell model Cell physiology Cells Degenerative Disorder Detection Development Diabetes Mellitus Disease Disease model Drug Formulations Ectoderm Embryo Endoderm Equipment Excision Flow Cytometry Genes Genetic Gold Heart Diseases Hip region structure Human Immunoblot Analysis Injury Lasers Lead Left Magnetism Measures Mesoderm Methods Modeling Modification Parkinson Disease Patients Peptide Phage Display Library Peptide antibodies Peptides Phage Display Pharmaceutical Preparations Phase Phenotype Plants Pluripotent Stem Cells Population Protocols documentation Reagent Reporting Reproducibility Residual state Reverse Transcriptase Polymerase Chain Reaction Risk Safety Simulate Small Business Innovation Research Grant Sorting - Cell Movement Source Stem Cell Research Stem cells Stress System Targeted Toxins Teratoma Testing Therapeutic Time Toxic effect Toxin Toxin Conjugates Transplantation Undifferentiated Western Blotting cell killing cell type clinical application cost effective cytotoxic cytotoxicity design drug discovery drug testing flexibility human stem cells immunogenic in vivo induced pluripotent stem cell killings particle physical separation public health relevance regenerative regenerative therapy screening stem tumor

项目摘要

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）：人类多能干（hPS）细胞作为可再生细胞来源具有巨大潜力，可用于药物发现、疾病建模和移植以替代因退行性疾病或损伤而损失的细胞。的一个关键障碍 hPS细胞研究的成功应用在于缺乏生产纯hPS细胞衍生物群体的方法。污染细胞既是药物发现的一个问题，在药物发现中需要纯群体来实现测定的重现性，又在移植中存在安全问题，因为它们有可能形成肿瘤或分化为不适当的细胞类型。具体来说，我们在此建议解决残留污染未分化 hPS 细胞的问题，这些细胞有可能形成畸胎瘤。我们建议开发 hPS 细胞靶向毒素 (hPS-CTT)，它可以杀死残留的 hPS 细胞，同时对分化细胞群的损害或改变最小。为了实现选择性细胞靶向毒性，我们将内化 hPS 选择性靶向剂与植物毒素皂草素结合起来，皂草素是仅在细胞内化时有毒。这种方法比之前提出的基因修饰和物理分离方法简单得多，这些方法需要进行可能改变或应激所需细胞群的细胞操作。它也比之前提出的细胞毒性抗体更加灵活，因为任何内化抗体或肽都可以适用于靶向细胞杀伤。此外，hPS-CTT 设计为与死亡细胞一起去除，从而减少其对存活细胞的潜在影响。为了证明第一阶段的可行性，我们将筛选用于选择性 hPS 细胞靶向的抗体和肽。细胞靶向剂 (CTA) 将间接与毒素结合，并测试其去除模型细胞系统中未分化 hS 细胞的能力，该模型细胞系统包含已分化 hPS 细胞和未分化 hPS 细胞的混合物。我们将通过流式细胞术、PluriTest 和定量 RT-PCR 测量治疗前后的 hPS 细胞含量。将使用体内畸胎瘤测定来测试最佳 hPS-CTT 去除 hPS 细胞的能力。 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测定来测量。将各种靶向剂与次级毒素缀合物结合起来的双组分系统将在第一阶段使用以显示可行性。在第二阶段，我们将开发直接与毒素结合的细胞靶向剂，并进一步优化 hPS 细胞去除方案。