Developing a platform for engineering customizable cell-cell signaling in vivo

抽象的。合成生物学结合了生物学和工程学原理来调节细胞 过程，并正在成为生物医学研究的一个重要领域。到目前为止，合成生物学已经 主要集中在操纵细胞内的过程，最显著的是控制基因表达或 新陈代谢，并将它们排列成执行离散功能的模块。相比之下，当前的电池- 基于文化的合成方法不适合操作控制交互的过程 在组织水平上创造所需的结果，这一能力将具有特殊的价值 在组织工程和再生医学领域。现在需要的是一种在基因上容易驯服的 VIVO平台，可在其中快速创建、测试、优化和 多样化，然后在系统中部署和进一步完善，具有治疗和 生物技术应用。我们建议通过建立果蝇系统来满足这一要求 用于设计控制组织行为的合成细胞间信号。我们已经开发出了样机 基于Notch激活的基本机制的合成配体/受体系统，其中 配体内吞施加的机械力诱导细胞外“力敏感”的裂解 受体的结构域。我们已经将我们的合成受体与一种新的遗传协议配对，以控制 配体/受体的相互作用，进行功能筛选和改变合成受体的输出。第一, 我们将通过进行大规模的新的筛选来使我们的合成受体曲目多样化 异源的力敏感切割结构域。其次，我们将严格评估我们的新受体 它们是否适合在电路中使用，并表征它们的重要响应参数，例如 信号的效力和调节的能力。最后，我们将组装合成信令电路 在上皮组织内产生可预测的输出的模块。如果成功，建议的 实验将是我们朝着建立一个易于处理的活体系统的长期目标迈出的重要一步 用于开发细胞-细胞信号技术，该技术在组织工程和 再生医学。

Abstract. Synthetic biology combines biological and engineering principles to regulate cellular processes, and is emerging as an important area of biomedical research. To date, synthetic biology has focused largely on manipulating processes inside cells, most notably to control gene expression or metabolism, and arranging them into modules that perform discrete functions. By contrast, current cell- culture based synthetic approaches are ill-equipped to manipulate processes that control interactions between cells to create desired outcomes at the tissue level, a capacity that would be of particular value in the fields of tissue engineering and regenerative medicine. What is needed is a genetically tractable in vivo platform within which synthetic cell-cell signaling tools can be rapidly created, tested, optimized and diversified, before they are deployed and further refined in systems that have therapeutic and biotechnological applications. We propose to fulfill this requirement by establishing a Drosophila system for designing synthetic intercellular signaling that controls tissue behavior. We have developed prototype synthetic ligand/receptor systems predicated on the basic mechanism of Notch activation, where mechanical force exerted by ligand endocytosis induces the cleavage of an extracellular “force-sensitive” domain of the receptor. We have paired our synthetic receptors with a new genetic protocol for controlling ligand/receptor interactions, conducting functional screens and altering synthetic receptor outputs. First, we will diversify our repertoire of synthetic receptors by conducting a large-scale screen for new heterologous force-sensitive cleavage domains. Second, we will rigorously assess our new receptors for their suitability to be used in circuits and characterize their important response parameters, such as the potency of signal and capacity for regulation. Last, we will assemble circuits of synthetic signaling modules to produce predictable outputs within an epithelial tissue. If successful, the proposed experiments will be a significant step toward our long-term goal of establishing a tractable in vivo system for developing cell-cell signaling technology that has future applications in tissue engineering and regenerative medicine.