De Novo Engineering of Small Molecule-Actuatable Biosensors for Cell Therapy

用于细胞治疗的小分子可驱动生物传感器的重新设计 摘要 此前的小分子药物和生物制剂、基于细胞的疗法，如嵌合抗原的使用 CAR-T细胞（CAR-T细胞）正在成为医学的新支柱。但相比 传统药物，由于与控制细胞相关的困难，它们更容易受到安全问题的影响。 在治疗环境中的行动。我的研究计划旨在开发一种新的方法来基因工程 用于时空控制细胞行为的编码生物传感器。我们关注的是 使用小分子控制细胞治疗反应、增殖、死亡、迁移 沟通和新陈代谢。我们的策略是设计化学诱导二聚化（CID）系统， 其中两种蛋白质仅在小分子存在下二聚化。到目前为止，只有少数CID系统 但是它们的二聚化诱导剂对于临床应用并不理想。CID系统的重新设计 对给定小分子的所需亲和力和特异性仍然是蛋白质领域中未解决的问题 工程.我们将通过将计算蛋白质设计与我们最近开发的 单分子相互作用测序（SMI-seq）技术。SMI-seq有可能破解一个密钥 通过实现大规模、二维（或“逐个文库”）筛选两个 CID结合物变体文库。我们将采用两种并行的方法来设计CID系统： 筛选计算设计的结合物文库，和ii）随机筛选极其多样的组合文库， 使用免疫球蛋白或从头设计的支架的结合文库（>109）。我们会评估成功率， 周转时间和成本效益的两种方法通过测试一套临床批准的抗病毒药物 作为CID诱导剂具有优异的细胞内递送效率的药物。最后，我们将演示如何使用 设计了生物传感器来控制培养细胞和小鼠模型中的细胞过程。成功 这项研究的完成将为工程化配体响应蛋白组装体开辟新的可能性， 蛋白质设计的未知领域设计的CID系统将显着扩大化学遗传 这是一个用于基因和细胞治疗以及系统和合成生物学研究的工具包。

De Novo Engineering of Small Molecule-Actuatable Biosensors for Cell Therapy ABSTRACT Preceded by small-molecule drugs and biologics, cell-based therapies, such as the use of chimeric antigen receptor T (CAR-T) cells against blood cancers, is becoming a new pillar of medicine. However, compared with traditional drugs, they are more susceptible to safety concerns due to difficulties associated with controlling cell actions in a therapeutic setting. My research program aims to develop a novel approach to engineer genetically encoded biosensors for spatiotemporal control of cell behaviors. We focus on the de novo engineering of the biosensors for using small molecules to control cell therapeutic responses, proliferation, death, migration, communication, and metabolism. Our strategy is to design chemically induced dimerization (CID) systems, in which two proteins dimerize only in the presence of a small molecule. To date, only a few CID systems are available and their dimerization inducers are not ideal for clinical use. The de novo design of CID systems with desired affinity and specificity for given small molecules remains an unsolved problem in the field of protein engineering. We will solve this problem by coupling computational protein design to our recently developed single-molecular-interaction sequencing (SMI-seq) technology. SMI-seq has the potential to break a key barrier to CID engineering by enabling large-scale, two-dimensional (or ‘library-by-library’) screening of two CID binder variant libraries. We will apply two parallel approaches to engineer CID systems: i) targeted screening of computationally designed binder libraries, and ii) random screening of vastly diverse combinatorial binder libraries (>109) using immunoglobulin or de novo designed scaffolds. We will assess the success rates, turnaround times, and cost-effectiveness of both approaches by testing a set of clinically approved antiviral drugs with excellent intracellular delivery efficiency as CID inducers. Finally, we will demonstrate the use of designed biosensors to control cellular processes in both cultured cells and a mouse model. Successful completion of this research will open up new possibilities for engineering ligand-responsive protein assemblies, an unexplored territory of protein design. Designed CID systems will significantly expand the chemogenetic toolkit for gene- and cell-based therapies, as well as systems and synthetic biology research.