Synthetic toolkit for precision gene expression control and signal processing in mammalian cells

用于哺乳动物细胞中精确基因表达控制和信号处理的合成工具包

• 批准号: 10153781
• 负责人: Ahmad Samir Khalil
• 金额: $ 66.15万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10153781
• 关键词: Address; Adoption; Advanced Development; Autoimmunity; Behavior; Biochemical; Biological; Biological Process; Biomedical Research; Cell Differentiation process; Cell Therapy; Cells; Cellular immunotherapy; Chemicals; Communities; Complex; Cues; Custom; Development; Disease; Dose; Drug Design; Engineering; FDA approved; Fostering; Gene Activation; Gene Expression; Generations; Genes; Genetic Transcription; Goals; Human; Human Biology; Human Engineering; Immune; Immunologist; Lead; Ligands; Malignant Neoplasms; Mammalian Cell; Methods; Modeling; Oncoproteins; Organoids; Output; Pharmaceutical Preparations; Physiology; Play; Process; Property; Publishing; Regenerative Medicine; Regulation; Reporter; Research Personnel; Role; Scheme; Science; Scientist; Signal Transduction; Specificity; Stimulus; System; Technology; Therapeutic; Tissue Engineering; Tissues; Transcriptional Regulation; Viral Vector; Work; Yeasts; base; cell behavior; cellular engineering; combinatorial; cytokine; design; flexibility; gene product; improved; information processing; interest; notch protein; novel; novel diagnostics; novel therapeutics; programs; response; signal processing; spatiotemporal; synthetic biology; therapy development; tool; transcription factor; tumor

PROJECT SUMMARY/ABSTRACT Cells activate precise gene expression programs in response to multifactorial chemical and biological stimuli. The purposeful manipulation of this process is a principal goal of synthetic biology, and its application to human cells could lead to breakthroughs in our understanding of human biology and in the development of next-generation diagnostics and therapeutics that respond in sophisticated ways to disease. Unfortunately, tools to artificially control gene expression in mammalian cells have significant limitations, constraining our ability to study fundamental biological processes and design more effective cell-based therapies. The most widely-used tools are older generation technology, derived from bacterial transcriptional systems. These are greatly limited in number, which restricts the number of gene products that can be simultaneously controlled. Additionally and importantly, they use “simple” one-to-one regulatory interactions, imposing fundamental restrictions on the regulatory flexibility and sophistication of designer systems. As a consequence, researchers are unable to create sophisticated gene expression controllers that can flexibly sense and integrate biochemical signals (e.g. ligands, chemical inducers, disease cues), and tune or reshape corresponding gene activation profiles. Among the many biomedical applications that would be transformed by these precision gene expression controllers in mammalian cells is the development of cell-based therapeutics for cancer, auto- immunity, and regenerative medicine, which can suffer from issues related to over-activation and tissue specificity. We propose to overcome these barriers by developing a novel synthetic toolkit for gene expression control in mammalian cells. Inspired by the natural design of metazoan transcriptional systems, our framework is based on synthetic transcription factors (synTFs) that can be programmed to assemble cooperatively in multivalent complexes. Our previous work showed that cooperative synTFs enable construction of gene expression control circuits with greatly expanded signal processing behavior in yeast. Here we will develop and characterize mammalian self-assembling synTFs that have superior properties for installation into human cells relative to existing tools. We will use these tools to develop three classes of gene expression controllers, which we will demonstrate in human immune cells, chosen for their important role in human physiology and their potential for cellular therapy: (1) Inducible controllers regulated by orthogonal, FDA-approved drugs. (2) Cell- autonomous controllers that sense and process biological stimuli, including ligand recognition by synthetic Notch receptors and microenvironmental cues. (3) Signal integration controllers that can perceive and integrate multiple biological signals to activate transcriptional programs. We anticipate that this toolkit will be broadly used by researchers to enable precision gene expression control across mammalian systems, including in biomedical applications of synthetic biology, cell reprogramming, and cell-based therapeutics. We will make our tools and design framework freely available to the academic scientific community.

项目概要/摘要 细胞激活精确的基因表达程序以响应多因素的化学和生物刺激。 有目的地操纵这一过程是合成生物学的主要目标，及其应用 人类细胞可能会导致我们对人类生物学的理解和发展的突破 以复杂的方式应对疾病的下一代诊断和治疗方法。 人工控制哺乳动物细胞基因表达的工具有很大的局限性，限制了我们的研究 研究基本生物过程和设计更有效的细胞疗法的能力。 广泛使用的工具是源自细菌转录系统的较旧技术。 数量上受到很大限制，限制了可以同时控制的基因产物的数量。 此外，重要的是，他们使用“简单”的一对一监管互动，强加基本的 因此，研究人员对设计系统的监管灵活性和复杂性进行了限制。 无法创建能够灵活感知和整合的复杂基因表达控制器 生化信号（例如配体、化学诱导物、疾病线索），并调整或重塑相应的基因 这些精度将改变许多生物医学应用。 哺乳动物细胞中的基因表达控制器正在开发基于细胞的癌症、自体 免疫和再生医学，可能会遇到与过度激活和组织相关的问题 我们建议通过开发一种新的基因表达合成工具包来克服这些障碍。 我们的框架受到后生动物转录系统自然设计的启发。 基于合成转录因子 (synTF)，可以对其进行编程以协作组装 我们之前的工作表明，协作性 synTF 能够构建基因。 我们将在酵母中开发和大大扩展信号处理行为的表达控制电路。 表征哺乳动物自组装 synTF，其具有安装到人类细胞中的优异特性 相对于现有的工具，我们将使用这些工具来开发三类基因表达控制器。 我们将在人类免疫细胞中进行演示，选择它们是因为它们在人类生理学中的重要作用及其 细胞治疗的潜力：(1) 由 FDA 批准的正交药物调节的诱导控制器 (2) Cell-。 感知和处理生物刺激的自主控制器，包括合成的配体识别 (3)可感知、集成的信号集成控制器 我们预计该工具包将广泛用于激活转录程序。 研究人员使用它来实现跨哺乳动物系统的精确基因表达控制，包括 我们将进行合成生物学、细胞重编程和基于细胞的疗法的生物医学应用。 我们的工具和设计框架免费提供给学术科学界。