Bottom-up, high-throughput prototyping of extracellular vesicle mimetics using cell-free synthetic biology

项目摘要 使用无细胞合成生物学的细胞外囊泡模拟物的自下而上的高通量原型设计 细胞分泌细胞外囊泡（EV），作为细胞间通讯的主要信使 并且被研究为有前景的药物递送载体和治疗剂。然而，本发明的临床应用 EV由于其低产量、杂质和固有的异质性而受到阻碍。原生EV包含 许多生物活性成分，如RNA和蛋白质，分布在许多亚群中。 这种生物复杂性既是天然电动汽车的优势，也是其致命弱点。虽然这其中的各种特征 尽管复杂性使得EV具有有益的治疗效果，但不清楚哪一种起主导作用。然而，在这方面， 蛋白质和RNA的复杂集合导致异质性EV，这对于研究和用作 标准化治疗。因此，从整体生物分子中分离出并定义关键的生物分子特征， 异质集将允许我们执行EV质量控制并可重复地生产或研究EV。一 寻找EV的关键分子部分的主要瓶颈是缺乏高通量方法。到 克服这个困难，我们的团队将创建一个基于合成生物学的，无细胞的高通量发现 平台该平台将能够使用无细胞合成方法合成EV模拟物（目标1）， 结合体外EV模拟物效力的高通量检测（目的2）。选择EV模拟物还将 使用神经保护和免疫调节的体内模型系统进行研究（目的3）。在整个 在本研究中，我们将使用天然间充质干细胞/基质细胞EV和神经系统疾病作为我们的模型系统 评估平台。我们的工作将使高通量研究电动汽车的任何疾病和生物学 感兴趣的问题此外，我们还将揭示电动汽车的新见解，解决电动汽车中的关键争论话题。 领域

PROJECT SUMMARY Bottom-up, high-throughput prototyping of extracellular vesicle mimetics using cell-free synthetic biology Cells secrete extracellular vesicles (EVs) that function as primary messengers of intercellular communication and are studied as promising drug-delivery vehicles and therapeutics. However, the clinical application of native EVs has been hindered by their low production yield, impurity, and inherent heterogeneity. Native EVs contain many biologically active components, such as RNAs and proteins, spread out over numerous subpopulations. This biological complexity is both the strength and the Achilles’ heel of native EVs. While various features of this complexity enable the beneficial therapeutic effects of EVs, it is not clear which plays a dominant role. However, the complex set of proteins and RNAs results in heterogeneous EVs that are challenging to study and use as a standardized treatment. Therefore, separating out and defining the critical biomolecular features from the overall heterogeneous set will allow us to perform quality control of EVs and to reproducibly produce or study EVs. A major bottleneck in finding the critical molecular parts of EVs is the lack of high-throughput methods. To overcome this difficulty, our team will create a synthetic biology-based, cell-free high-throughput discovery platform. The platform will be able to synthesize EV mimetics using a cell-free synthesis approach (Aim 1), coupled with high-throughput examination of EV mimetic potency in vitro (Aim 2). Select EV mimetics will also be investigated using an in vivo model system of neuroprotection and immune modulation (Aim 3). Throughout the study, we will use native mesenchymal stem/stromal cell EVs and neurological diseases as our model system to evaluate the platform. Our work will enable the high-throughput study of EVs for any disease and biological questions of interest. In addition, we will unveil new insights into EVs that address key debated topics in the EV field.