Cell targeting with synthetic sense-and-respond protease circuits

使用合成的感知和响应蛋白酶电路进行细胞靶向

• 批准号: 10447755
• 负责人: MICHAEL B ELOWITZ
• 金额: $ 60.24万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10447755
• 关键词: Address; Animals; Biological Assay; Cancer cell line; Cell Death; Cell model; Cells; DNA; Devices; Disease; Dosage Compensation (Genetics); Engineering; Ensure; Foundations; Future; Gene Dosage; Genome; Hepatocyte; Logic; Malignant Epithelial Cell; Malignant Neoplasms; Messenger RNA; Modeling; Modification; Normal Cell; Oncogenic; PI3K/AKT; Pathway interactions; Peptide Hydrolases; Play; Population; Primary carcinoma of the liver cells; Process; Protein Binding Domain; Protein Engineering; Proteins; Reporter; Research; Role; Signal Transduction; Specificity; System; Tertiary Protein Structure; Testing; Therapeutic; Transcript; Variant; Viral; Work; base; cancer cell; cell killing; cell type; cellular engineering; combinatorial; design; design and construction; dosage; experimental study; flexibility; hepatocellular carcinoma cell line; intein; lipid nanoparticle; liver cancer model; mRNA delivery; mathematical model; nanobodies; neoplastic cell; operation; programs; protein expression; ras Oncogene; reconstitution; response; sensor; signal processing; synthetic biology; synthetic protein

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.

通过合成感官和响应蛋白酶电路进行细胞靶向 抽象的 生物医学中的一个基本挑战是开发可以针对特定细胞群体的治疗剂。 基于彼此相互作用并与内源性相互作用的工程蛋白质的合成蛋白回路 蜂窝路径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 感知关键的蜂窝路径，处理该信息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反应。合成蛋白质电路也可能是 编码为mRNA并暂时传递，以避免基因组修饰。我们最近表明病毒 可以设计蛋白酶以模块化的方式相互调节，从而构建多样 蛋白质级的功能来自有限数量的组件。但是，仍然存在关键挑战：可以 将工程蛋白酶电路推广，以感知多种癌症途径，而对 细胞？他们可以实现更广泛的信号处理功能，包括阈值， 集成和剂量补偿以允许在各种细胞环境中使用多功能和精确的功能？和， 当瞬时传递为mRNA时，它们可以选择性地靶向癌细胞吗？在这里，我们旨在发展这个 通过创建新的途径感应能力，进入靶向癌细胞的更广泛平台， 设计灵活的信号处理模块，并展示具有感知和杀死特定目标细胞的能力 类型。我们将专注于肝细胞癌（HCC）的细胞模型，这种疾病仍然存在 挑战性治疗，但对mRNA递送是相对允许的。在AIM 1中，我们将设计和验证 在HCC中起关键作用的主要致癌途径的蛋白酶传感器。这些传感器有条件 响应靶蛋白的定位，聚类，活性或丰度来激活病毒蛋白酶。在 AIM 2，我们将创建基于蛋白酶的电路模块，以积极处理这些信号。我们将设计 阈值模块以抑制对正常细胞基础途径活动的不希望反应，并 组合逻辑模块可以允许从不同传感器中的信号集成。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AIM 3中，我们 将设计有选择性地杀死HCC细胞系的mRNA传递电路，对正常的影响很小 肝细胞。该研究计划将建立mRNA递送蛋白酶的端到端可行性 电路并为将来的基于可编程电路的治疗学提供基础。