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去新工程 摘要 在小分子药物和生物制品、基于细胞的治疗之前，如使用嵌合抗原 受体T细胞(CAR-T)抗血癌，正在成为医学的新支柱。然而，与 传统药物，由于控制细胞的困难，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进行基因工程 用于细胞行为时空控制的编码生物传感器。我们专注于从头开始的工程 生物传感器用于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(CID)系统， 其中两种蛋白质只有在小分子存在的情况下才能二聚化。到目前为止，只有少数几个CID系统 但它们的二聚化诱导剂并不适合临床使用。CID系统的从头设计 对于给定的小分子所需的亲和力和特异性在蛋白质领域仍然是一个未解决的问题 工程学。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有可能破解密钥 通过实现大规模、二维(或逐个库)的两个 CID结合变异库。我们将应用两种并行方法来设计CID系统：i)目标 筛选计算设计的粘结剂文库，以及ii)随机筛选差异很大的组合 结合文库(&gt；109)使用免疫球蛋白或从头设计的支架。我们将评估成功率， 通过测试一组临床批准的抗病毒药物来测试两种方法的周转时间和成本效益 具有良好细胞内给药效率的药物作为CID诱导剂。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设计配体反应蛋白组件开辟新的可能性， 蛋白质设计的一个未被探索的领域。设计的CID系统将显著扩大化学发生 用于基因和细胞疗法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.