Cell targeting with synthetic sense-and-respond protease circuits

用合成的感测-应答蛋白酶电路靶向细胞 摘要 生物医学的一个基本挑战是开发可以靶向特定细胞群的治疗方法。 合成蛋白质电路，基于工程蛋白质，相互作用，并与内源性 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 感知关键的细胞通路，处理这些信息以分类细胞状态，并通过以下方式做出反应： 有条件地触发细胞死亡或其它有益的反应。合成蛋白质电路也可能是 编码为mRNA并瞬时递送以避免基因组修饰。我们最近发现， 蛋白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酶。 蛋白质水平的功能来自有限数量的组分。然而，关键的挑战仍然存在： 工程化的蛋白酶电路可以被推广到以最小的扰动感测多种癌症途径， 细胞？它们能否实现更广泛的信号处理功能，包括阈值处理， 整合和剂量补偿，以允许在不同的细胞环境中的多功能和精确的功能？而且， 当它们以mRNA的形式瞬时传递时，是否可以选择性地靶向癌细胞？在这里，我们的目标是开发这个 通过创造新的通路传感能力， 设计灵活的信号处理模块，并展示检测和杀死特定靶细胞的能力 类型我们将重点关注肝细胞癌（HCC）的细胞模型， 治疗具有挑战性，但相对允许mRNA递送。在目标1中，我们将设计和验证 主要致癌途径的蛋白酶传感器在HCC中发挥关键作用。这些传感器有条件地 响应于靶蛋白的定位、聚集、活性或丰度而激活病毒蛋白酶。在 目标2，我们将创建基于蛋白酶的电路模块，积极处理这些信号。我们将工程师 阈值模块，用于抑制对正常细胞中的基础途径活性的不期望的响应，以及 组合逻辑模块，以允许来自不同传感器的信号的与式积分。此外，我们将 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mRNA传递电路，选择性地杀死HCC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mRNA传递蛋白酶的端到端的可行性 并为未来基于可编程电路的治疗提供基础。

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.