Cell targeting with synthetic sense-and-respond protease circuits

利用合成的正义和反应蛋白酶回路进行细胞靶向 摘要 生物医学的一个根本挑战是开发能够针对特定细胞群体的治疗方法。 合成蛋白质回路，基于相互作用和与内源性相互作用的工程蛋白质 细胞通路，可以提供一个强大的平台来应对这一挑战。这些电路可以直接 感知关键细胞通路，对信息进行处理以对细胞状态进行分类，并通过 有条件地触发细胞死亡或其他有益的反应。合成蛋白质电路也可能是 编码为信使核糖核酸，并瞬时传递，以避免基因组修改。我们最近展示了病毒 蛋白水解酶可以被设计成以模块化的方式相互调节，从而允许构建不同的 蛋白质水平的功能来自有限数量的成分。然而，关键挑战仍然存在：能否 工程化的蛋白酶电路被推广到检测多个癌症途径，并将扰动降至最低 牢房？他们能否实现更广泛的信号处理功能，包括阈值处理、 集成和剂量补偿，以在不同的细胞环境中实现多功能和精确的功能？和, 当它们以信使核糖核酸的形式瞬时传递时，它们能选择性地靶向癌细胞吗？在这里，我们的目标是开发这一点 通过创建新的路径感知功能，将系统整合为更广泛的靶向癌细胞的平台， 设计灵活的信号处理模块，并演示感知和杀死特定目标细胞的能力 类型。我们将关注肝细胞癌的细胞模型，这是一种残留的疾病 治疗具有挑战性，但对信使核糖核酸的传递相对宽松。在目标1中，我们将设计和验证 在肝细胞癌中起关键作用的主要致癌途径的蛋白水解酶传感器。这些传感器是有条件的 激活病毒蛋白酶，以响应目标蛋白的定位、聚集、活性或丰度。在……里面 目标2，我们将创建基于蛋白酶的电路模块，这些模块可以主动处理这些信号。我们将进行工程设计 阈值模块，用于抑制正常细胞对基础通路活动的不良反应，以及 组合逻辑模块，允许对来自不同传感器的信号进行AND类集成。此外，我们还将 设计剂量补偿模块，使蛋白质表达对电路传递不敏感，在目标3中，我们 将设计信使核糖核酸传递的电路，选择性地杀死肝癌细胞系，对正常细胞的影响最小 肝细胞。这项研究计划将建立信使核糖核酸蛋白酶的端到端的可行性。 并为未来基于电路的可编程疗法奠定了基础。

Cell targeting with synthetic sense-and-respond protease circuits Abstract A fundamental challenge in biomedicine is developing therapeutics that can target specific cell populations. Synthetic protein circuits, based on engineered proteins that interact with one another and with endogenous cellular pathways, could provide a powerful platform to address this challenge. These circuits could directly sense key cellular pathways, process that information to classify the cellular state, and respond by conditionally triggering cell death or other beneficial responses. Synthetic protein circuits could also be encoded as mRNA and delivered transiently to avoid genome modification. We recently showed that viral proteases could be engineered to regulate one another in a modular way, allowing construction of diverse protein-level functions from a limited number of components. However, key challenges remain: Can engineered protease circuits be generalized to sense multiple cancer pathways with minimal perturbations to the cell? Can they implement a broader set of signal processing capabilities, including thresholding, integration, and dosage compensation to allow for versatile and precise function in diverse cell contexts? And, can they selectively target cancer cells when transiently delivered as mRNA? Here, we aim to develop this system into a broader platform for targeting cancer cells by creating new pathway sensing capabilities, designing flexible signal processing modules, and demonstrating the ability to sense and kill specific target cell types. We will focus on cellular models of hepatocellular carcinoma (HCC), a disease which remains challenging to treat but is relatively permissive for mRNA delivery. In Aim 1, we will design and validate protease sensors of major oncogenic pathways that play critical roles in HCC. These sensors conditionally activate viral proteases in response to the localization, clustering, activity, or abundance of target proteins. In Aim 2, we will create protease-based circuit modules that actively process these signals. We will engineer thresholding modules to suppress undesired responses to basal pathway activities in normal cells, and combinatorial logic modules to allow AND-like integration of signals from distinct sensors. In addition, we will design dosage compensation modules that make protein expression insensitive to circuit delivery, In Aim 3, we will design mRNA-delivered circuits that selectively kill HCC cell lines with minimal impact on normal hepatocytes. This research program will establish the end-to-end feasibility of mRNA-delivered protease circuits and provide the foundations for future programmable circuit-based therapeutics.