A synthetic signaling pathway engineering platform for creating precision cell-based sense-and-response devices

摘要 蛋白质信号网络被细胞用来感知、处理和响应物理和分子特征 在他们的外部环境中。膜受体介导的人工信号网络工程 感知与疾病相关的信号对治疗反应的反应可能导致发展的突破 能够自主检测和治疗疾病的动态细胞治疗设备。相比之下， 对于依赖磷酸化来转导外部信号的本地信令网络，目前的方法 在人类中构建合成的信号网络依赖于非本地的调节机制并运行 在缓慢的时间尺度上或通过单周转事件。因此，构建意义和- 响应计划，准确地将环境波动与输出响应相结合，或者可以灵活地- 公司不同的受体介导的输入。设计基于磷酸化的感觉和反应的能力 程序可以实现类似于本地路径的功能行为，包括快速检测和整合 细胞外信号。通过在不同的细胞外环境之间实现微调的区分，例如 这些计划可以提高基于细胞的治疗的安全性和有效性。在这个项目中，我们将建立一个 在人类细胞中设计基于合成磷酸化的信号转导的通用方法，奠定了一个基础- 能够感知与疾病相关的分子线索的下一代细胞治疗设备的基础，以及 将它们转化为定量定义的治疗反应。为了实现我们的目标，我们将利用 我们实验室最近开发的合成生物学平台，可以自下而上地构建合成磷酸盐- 使用工程信号蛋白的磷酸化电路。正如我们的初步工作所表明的，这个平台 可以用来创建合成信号通路，连接受体介导的胞外分子检测- 与下游细胞过程(如转录)的激活有关。在这里，我们将调查这个平台是否 可用于设计感知和反应程序来治疗炎症性疾病。具体来说，我们将：1) 展示合成通路与感知不同生物分子信号的受体连接的能力 与炎症有关；2)设计信号网络，整合多种信号并做出反应，但不包括 在存在明确的炎症信号组合的情况下；3)间充质的测试途径 基质细胞(MSCs)来评估我们平台的可译性。我们的工作将阐明以下基本原则 设计合成信令电路并提供强大的技术平台，以创建定制的 感知和反应程序，可以精确区分健康和疾病组织的特征。在……里面 除了疾病监测和诊断应用，这些基于细胞的精密治疗设备可以 用于治疗各种疾病，从炎症和自身免疫疾病，到组织创伤和癌症。

SUMMARY Protein signaling networks are used by cells to sense, process, and respond to physical and molecular features in their external environment. Engineering artificial signaling networks that couple membrane receptor-mediated sensing of disease-associated signals to therapeutic responses could lead to breakthroughs in the development of dynamic cell-based therapeutic devices capable of autonomously detecting and treating disease. In contrast to native signaling networks, which rely on phosphorylation to transduce external signals, current approaches for constructing synthetic signaling networks in humans rely on nonnative regulatory mechanisms and operate on slow timescales or via single-turnover events. As a consequence, it is challenging to construct sense-and- respond programs that accurately couple environmental fluctuations to output response, or that can flexibly in- corporate diverse receptor-mediated inputs. The ability to engineer phosphorylation-based sense-and-response programs could enable functional behavior resembling native pathways, including rapid detecting and integration of extracellular signals. By enabling fine-tuned discrimination between different extracellular environments, such programs could enhance safety and efficacy profiles of cell-based therapies. In this project, we will establish a generalizable approach for engineering synthetic phosphorylation-based signaling in human cells, laying a foun- dation for next-generation cell therapy devices capable of sensing molecular cues associated with disease, and converting them into quantitatively defined therapeutic responses. To accomplish our goals, we will leverage a synthetic biology platform recently developed by our lab that enables bottom-up construction of synthetic phos- phorylation circuitry using engineered signaling proteins. As our preliminary work demonstrates, this platform can be used to create synthetic signaling pathways connecting receptor-mediated detection of extracellular mol- ecules to activation of downstream cellular processes (e.g., transcription). Here, we will investigate if this platform can be used to engineer sense-and respond program to treat inflammatory disease. Specifically, we will: 1) demonstrate the ability of synthetic pathways to be wired to receptors that sense diverse biomolecular cues associated with inflammation; 2) engineer signaling networks that integrate multiple signals and respond exclu- sively in the presence of defined combinations of inflammatory cues and; 3) test pathways in mesenchymal stromal cells (MSCs) to assess translatability of our platform. Our work will illuminate foundational principles for engineering synthetic signaling circuits and deliver a powerful technology platform for creating customized sense-and-respond programs that can precisely distinguish between features of healthy and diseased tissue. In addition to disease monitoring and diagnostic applications, these precision cell-based therapy devices could be used treat diseases ranging from inflammatory and autoimmune disorders, to tissue trauma and cancer.