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

摘要 哺乳动物合成生物学的目标是用合成分子合理地规划细胞的行为 电路，它为不同的生物医学领域带来了巨大的希望，如细胞命运重新编程和肿瘤溶解 病毒学。合成电路主要是用转录因子构建的，并在 基于DNA的载体，与转录调控兼容，但可能插入和突变 宿主基因组。蛋白质水平的电路可能会运行得更快，在亚细胞中并行计算 隔室，并直接与细胞内源性输入/输出接口。它们还将使 开发基于RNA的载体，具有较低的突变风险，因为蛋白质水平的电路可以作为 这两种载体都可以正常工作，即使是从RNA载体表达的，也可以作为RNA的控制器 通过调节基本的病毒蛋白来感染病毒。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不同于转录单位， 缺乏可组合性(为不同任务选择和组装不同的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水解酶作为组成成分 蛋白质水平的电路，并展示了广泛的各种功能。在最初的成功基础上，我将 增强蛋白酶回路的能力，同时开发用于其递送的RNA载体。为了更好地 将蛋白酶电路耦合到细胞内源性输入/输出，我将构建检测模块，将特定的 蛋白质的存在和信号事件转化为蛋白酶活性，以及破坏的执行模块 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 正交蛋白水解酶，并将其改造成可受其他蛋白水解酶调节。同时，为了方便 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，我将建立一个用于模拟蛋白酶电路的计算框架。至于送货，我会的 利用蛋白水解酶回路调节病毒必需蛋白，将负链RNA病毒改造成载体 以实现安全性和细胞类型的特异性。我还将测试载体载货能力的极限，并探索 提高限额的策略。总而言之，我的项目将产生一个更强大的平台来构建和 将不含DNA的合成电路送入哺乳动物细胞。 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，我将继续接受- 来自Elowitz博士和我们的合作者的深入的定量和数学培训，并获得关键 来自我的顾问的实验技术。我还将演示基本设计的可行性 在每个目标的背后。我训练有素的技能，以及个人设计，将在R00阶段结合在一起，产生 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。

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.