Advancing stem cell technologies using engineered zinc finger proteins

描述 摘要： 胚胎干细胞（ESC）和诱导多能干细胞（iPSC）具有无限复制和保持多能性的能力，为开发基于再生医学的新型疗法提供了巨大的机会。特别是iPSC技术可以提供开发个性化治疗策略的手段，其中患者自身的细胞可以被修饰并用于治疗他们的疾病而不需要免疫抑制。患者来源的ESC和iPSC还将为建模和研究各种疾病的生物学提供至关重要的试剂。尽管最近取得了巨大的进展，但在ESC和iPSC可常规用于治疗之前，仍有许多重大挑战有待解决。例如，用于产生iPSC的方法目前是低效的，具有相当大的改进空间。此外，用于产生特定突变的同源重组介导的基因靶向以低效率工作并且难以进行，特别是在人ESC和iPSC中。最后，用于将ESC和iPSC稳健地分化成感兴趣的细胞类型的方法通常是低效的，并且产生的细胞不能表现出所有期望的特征。该提案的目标是采用工程化锌指技术来解决这些阻碍ESC和iPSC研究和治疗应用的主要挑战。具体而言，该提案将利用PI在工程化锌指技术方面的专业知识来解决干细胞生物学和研究的三个重要挑战：（1）开发更有效的iPSC生成方法，（2）在人类ESC和iPSC中实现高效ZFN诱导的基因靶向，以及（3）创建更有效的方法将干细胞分化为所需的细胞类型。

DESCRIPTION Abstract: Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their abilities to replicate indefinitely and maintain pluripotency, offer tremendous opportunities for the development of novel regenerative medicine-based therapies. iPSC technology, in particular, may provide the means to develop personalized therapeutic strategies in which a patient's own cells might be modified and used to treat their disease without the need for immunosuppression. Patient-derived ESCs and iPSCs will also provide critically important reagents for modeling and studying the biology of various diseases. Despite tremendous recent progress, many significant challenges remain to be addressed before ESCs and iPSCs can be used routinely for therapeutics. For example, methods for generating iPSCs are currently inefficient with considerable room for improvement. In addition, homologous recombination-mediated gene targeting for creating specific mutations works with low efficiency and is difficult to perform, particularly in human ESCs and iPSCs. Lastly, methods for robustly differentiating ESCs and iPSCs into cell types of interest are typically inefficient and yield cells that fail to exhibit all desired characteristics. The goal of this proposal is to employ engineered zinc finger technology to address these major challenges impeding research and therapeutic applications of ESCs and iPSCs. Specifically, this proposal will utilize the PI's expertise in engineered zinc finger technology to address three important challenges for stem cell biology and research: (1) developing more efficient methods for iPSC generation, (2) enabling high efficiency ZFN-induced gene targeting in human ESCs and iPSCs, and (3) creating more efficient methods for differentiating stem cells into desired cell types of interest.