Advancing stem cell technologies using engineered zinc finger proteins

描述 摘要： 胚胎干细胞(ESCs)和诱导多能干细胞(IPSCs)具有无限复制和维持多能性的能力，为以再生医学为基础的新疗法的发展提供了巨大的机会。特别是，IPSC技术可以提供开发个性化治疗策略的手段，在这种策略中，患者自己的细胞可以被修改并用于治疗他们的疾病，而不需要免疫抑制。患者来源的ESCs和IPSCs也将为各种疾病的生物学建模和研究提供至关重要的试剂。尽管最近取得了巨大的进展，但在ESCs和IPSCs可以常规用于治疗之前，仍有许多重大挑战需要解决。例如，产生IPSC的方法目前效率低下，有相当大的改进空间。此外，同源重组介导的基因靶向产生特定突变的工作效率低且难以执行，特别是在人类ESCs和IPSCs中。最后，将ESCs和IPSCs有力地区分为感兴趣的细胞类型的方法通常效率低下，产生的细胞无法表现出所有所需的特征。这项提案的目标是利用工程锌指技术来解决这些阻碍ESCs和IPSCs研究和治疗应用的主要挑战。具体地说，这项建议将利用PI在工程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ESCs和IPSCs中实现高效的ZFN诱导的基因靶向，以及(3)创造更有效的方法将干细胞区分为所需的感兴趣细胞类型。

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.