Engineering Synthetic Receptor Systems That Can Detect Specific Cell-Cell Contact

 描述（由申请人提供）：合成生物学的目标之一是能够设计能够感知和报告新信号的细胞。可以被修饰以感测特定细胞-细胞接触的细胞对于广泛的生物医学和研究应用将是非常有用的，范围从细胞连接网络的映射到控制用于组织修复或再生的干细胞分化的确切位置。在初步研究中，我们最近开发了一种新的合成受体结构，基于Notch受体，其中我们可以交换由受体感知的细胞外配体。该受体在通过与表达同源配体（但不可溶性配体）的细胞接触而被激活时，诱导用户定义的转录应答。因此，这种工程化受体平台原则上可用于将各种细胞接触刺激灵活地连接到灵活的模块化反应。我计划描述和展示这个受体平台的两个应用。在K99阶段，我将实现两个目标。一个是通过表征其模块性来优化和机械地表征合成的细胞-细胞接触受体系统。为此，我将 利用蛋白质工程技术构建受体变异体库，通过在成纤维细胞中表达这些库来表征受体，并通过荧光激活细胞分选仪（FACS）监测报告基因的转录。第二个目标是使用这个合成受体平台作为工具包来绘制细胞-细胞连接。为此，我将使用第一个目标的文库，并通过活体显微镜观察报告基因的接触依赖性激活。我还计划通过实现逻辑门和对受体反应的时间控制来扩展系统的组合能力。对于R 00阶段，我计划开发第三个目标：使用这种合成受体平台作为控制细胞分化的一种方式，以响应成核细胞 或表面支架。我将用嵌合受体改造小鼠胚胎干细胞，使其只有在与邻居或支架提供的配体接触时才能分化。这些研究将为设计新一波强大的工具奠定基础，这些工具通过细胞-细胞和细胞-表面接触来控制细胞行为。通过这些受体报告和预测细胞行为的可能性将为基础研究、细胞治疗和组织工程建立新的范例。该受体平台的成功表征应该为我们提供一个可定制且高度灵活的工具包来控制细胞行为，并绘制活组织中细胞之间的复杂关系。在未来，我们将能够报告复杂的细胞-细胞接触，并根据它们在体内、神经元和其他发育环境中的邻居关系来区分细胞群。这些工具也将使复杂的胚胎干细胞分化程序的控制，无论是在体外增加我们的基本理解，并在体内控制干细胞分化的组织修复或再生的确切位置，也许专门响应与组织损伤或损伤相关的信号。

 DESCRIPTION (provided by applicant): One of the goals of synthetic biology is to be able to engineer cells that can sense and report on new signals. Cells that could be modified to sense specified cell-cell contacts would be extremely useful for a wide range of biomedical and research applications, ranging from mapping of cell connectivity networks to controlling the exact location of stem cell differentiation for tissue repair or regeneration. In preliminary studis, we have recently developed a new synthetic receptor construct, based on the Notch receptor, in which we can swap the extracellular ligands that are sensed by the receptor. This receptor, when activated by contact with a cell expressing the cognate ligand (but not soluble ligand), induces a user-defined transcriptional response. Thus this engineered receptor platform can in principle be used to flexibly link a wide variety of cell-contact stimuli to a flexible, modular response. I plan to characterize and show two applications of this receptor platform. During the K99 phase I will carry out two aims. One is to optimize and mechanistically characterize the synthetic cell-cell contact receptor system, by characterizing its modularity. For this aim, I will use protein- engineering techniques to build libraries of receptor variants; I will characterize th receptor by expressing these libraries in fibroblasts cells and monitor reporter transcription by fluorescent activation cell sorter (FACS). The second aim is to use this synthetic receptor platform as a toolkit to map cell-cell connectivity. For this aim, I will use the libraries of the irst aim and follow the contact-dependent activation of reporter genes by live microscopy. I plan also to extend the combinatorial power of the system by implementing logic gate and temporal control over receptor response. For the R00 phase, I plan to develop a third aim: use this synthetic receptor platform as a way to control cell differentiation in response to nucleating cell or surface scaffold. I will engineer mouse embryonic stem cells with chimeric receptors to differentiate only when contacted by ligands presented by neighbors or by scaffold. These studies will represent a foundation for the design of a new wave of powerful tools for the control of cell behavior via cell-cell and cell-surface contacts. The possibility of reporting and controllng cell behavior through these receptors will establish new paradigms for basic research, cell therapy and tissue engineering. The successful characterization of this receptor platform should give us a customizable and highly flexible toolkit to control cell behavior, as well as to map complex relationships between cells in live tissue. In the future we will be able to report on complex cell-cell contacts and distinguish populations of cells based on their neighbor relationships in vivo, in neurons and other developmental contexts. These tools will also enable the control of complex differentiation programs of ES cells both in vitro to increase our fundamental understanding, and in vivo to control the exact location of stem cell differentiation for tissue repair or regeneration, perhaps responding specifically to signals associated with tissue damage or injury.