Synthetic DNA-free Circuits for “Scarless” Programming of Mammalian Cells

用于哺乳动物细胞“无痕”编程的合成无 DNA 电路

• 批准号: 10379933
• 负责人: Xiaojing J Gao
• 金额: $ 24.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-10 至 2024-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10379933
• 关键词: Ablation; Adopted; Apoptosis; Basic Science; Behavior; Binding; Bioinformatics; Biotechnology; Cells; Cicatrix; Complex; Coupling; DNA; DNA delivery; Detection; Development; Dimerization; Elements; Engineering; Epitopes; Event; Foundations; Future; Genetic; Genetic Transcription; Genome; Genomics; Goals; Human; Immune response; Individual; Lead; Malignant Neoplasms; Mammalian Cell; Mathematics; Medical; Molecular; Oncogene Activation; Oncolytic; Outcome; Output; Pathologic; Patients; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phosphopeptides; Process; Proteins; Proteolysis; Public Health; RNA; RNA Viruses; Regulation; Regulation of Proteolysis; Research; Research Personnel; Risk; Running; Safety; Signal Transduction; Specificity; Techniques; Testing; Therapeutic; Training; Transcriptional Regulation; Viral; Viral Genome; Viral Proteins; Viral Vector; Virus; Virus Replication; base; cancer cell; career; cell behavior; cell type; computer framework; delivery vehicle; design; improved; in vivo; knock-down; nanobodies; operation; parallel computer; programs; protein aminoacid sequence; protein expression; ras Oncogene; reconstitution; skills training; small molecule; success; synthetic biology; synthetic construct; transcription factor; transmission process; ubiquitin-protein ligase; vector; virology

Abstract Mammalian synthetic biology aims to rationally program the behavior of cells with synthetic molecular circuits, and it holds great promises for diverse biomedical fields such as cell fate reprogramming and oncolytic virology. Synthetic circuits have been predominantly constructed with transcription factors, and delivered on DNA-based vectors that are compatible with transcriptional regulation but may insert into and mutagenize the host genome. Protein-level circuits would potentially operate faster, compute in parallel in subcellular compartments, and interface directly with cell endogenous inputs/outputs. They would also enable the development of RNA-based vectors with lower mutagenic risks, because protein-level circuits can serve as both cargos that functions properly even when expressed from an RNA vector, and as controllers for RNA viruses through regulation of essential viral proteins. However, despite researchers' efforts, protein circuits have been limited to a few ad hoc examples, because existing protein components, unlike transcriptional units, lack composability (the ability to select and assemble modular building blocks differently for different tasks). In our preliminary study, we successfully engineered viral proteases as composable elements for protein-level circuits, and demonstrated a broad variety of functions. Building upon the initial success, I will enhance the capability of protease circuits, and concurrently develop a RNA vector for their delivery. To better couple protease circuits to cell endogenous inputs/outputs, I will build detection modules that converts specific proteins' presence and signaling events into protease activity, and execution modules that knock down endogenous proteins. To meet a larger input/output palette with expanded computing capacity, I will mine more orthogonal proteases and engineer them to be regulatable by other proteases. Meanwhile, to facilitate the optimization of more complex circuits and to explore its unique features compared to traditional transcriptional circuits, I will establish a computational framework for simulating protease circuits. As for delivery, I will engineer a negative-strand RNA virus into a vector by regulating essential viral proteins with protease circuits to achieve safety and cell-type specificity. I will also test the limit of the vector's cargo capacity and explore strategies to raise the limit. All told, my project will engender a more powerful platform for constructing and delivering DNA-free synthetic circuits into mammalian cells. My career goal is to lead my independent research group devoted to establishing a general-purpose toolkit for non-mutagenic manipulation of mammalian cells. During the K99 phase, I will continue to receive in- depth quantitative and mathematical training from Dr. Elowitz and our collaborators, and acquire key experimental techniques from my consultants. I will also have demonstrated the feasibility of the basic designs behind each aim. My trained skills, as well as individual designs, will come together in the R00 phase, give rise to more complex circuits with direct biomedical relevance, and lay the foundation for my future career.

抽象的 哺乳动物合成生物学旨在利用合成分子合理地编程细胞的行为 它在细胞命运重编程和溶瘤等不同生物医学领域具有广阔的前景 病毒学。合成电路主要是用转录因子构建的，并传递到 基于 DNA 的载体，与转录调控兼容，但可能插入并诱变 宿主基因组。蛋白质级电路可能会运行得更快，在亚细胞中并行计算 室，并直接与细胞内源性输入/输出接口。他们还将使 开发具有较低诱变风险的基于 RNA 的载体，因为蛋白质水平电路可以充当 即使从 RNA 载体表达，这两种货物也能正常发挥作用，并作为 RNA 的控制器 病毒通过调节必需的病毒蛋白。然而，尽管研究人员做出了努力，蛋白质电路 仅限于几个特别的例子，因为现有的蛋白质成分与转录单位不同， 缺乏可组合性（针对不同任务以不同方式选择和组装模块化构建块的能力）。 在我们的初步研究中，我们成功地将病毒蛋白酶设计为可组合元件 蛋白质级电路，并展示了多种功能。在初步成功的基础上，我将 增强蛋白酶电路的能力，同时开发用于递送它们的RNA载体。为了更好 将蛋白酶电路与细胞内源性输入/输出耦合，我将构建将特定的 蛋白质的存在和蛋白酶活性的信号事件，以及击倒的执行模块 内源性蛋白质。为了满足更大的输入/输出调色板和扩展的计算能力，我将挖掘更多 正交蛋白酶并将其改造为可被其他蛋白酶调节。同时，为方便 优化更复杂的电路并探索其与传统转录相比的独特功能 电路，我将建立一个模拟蛋白酶电路的计算框架。至于送货，我会 通过用蛋白酶电路调节必需的病毒蛋白，将负链 RNA 病毒设计成载体 以实现安全性和细胞类型特异性。我也会测试载体的载货能力极限并探索 提高限制的策略。总而言之，我的项目将产生一个更强大的平台来构建和 将无 DNA 的合成电路传递到哺乳动物细胞中。 我的职业目标是领导我的独立研究小组致力于建立一个通用目的 用于哺乳动物细胞非诱变操作的工具包。在K99阶段，我将继续收到- 来自 Elowitz 博士和我们的合作者的深度定量和数学培训，并获得关键 我的顾问的实验技术。我还将展示基本设计的可行性 每个目标的背后。我训练有素的技能，以及个人设计，将在R00阶段汇集在一起​​，产生 到与生物医学直接相关的更复杂的电路，并为我未来的职业生涯奠定基础。

项目成果

Protease-controlled secretion and display of intercellular signals.

蛋白酶控制的细胞间信号的分泌和显示。

• 发表时间: 2022-02-17
• 影响因子: 16.6
• 作者: Vlahos, Alexander E;Kang, Jeewoo;Aldrete, Carlos A;Zhu, Ronghui;Chong, Lucy S;Elowitz, Michael B;Gao, Xiaojing J
• 通讯作者: Gao, Xiaojing J

Modular, programmable RNA sensing using ADAR editing in living cells.

在活细胞中使用 ADAR 编辑的模块化、可编程 RNA 传感。

• 发表时间: 2023-04
• 影响因子: 46.9
• 作者: Kaseniit, K Eerik;Katz, Noa;Kolber, Natalie S;Call, Connor C;Wengier, Diego L;Cody, Will B;Sattely, Elizabeth S;Gao, Xiaojing J
• 通讯作者: Gao, Xiaojing J