Synthetic Genetic Controller Circuits to Reprogram Cell Fate

用于重新编程细胞命运的合成遗传反馈控制器电路 PI：Domitilla Del Vecchio1；4 合作伙伴：詹姆斯·J·柯林斯2；4；5；6，托尔斯滕·施莱格7，罗恩·韦斯2；3；4 麻省理工学院机械工程系；麻省理工学院生物工程系 麻省理工学院电气工程与计算机科学系 4麻省理工学院合成生物学中心；5麻省理工学院和哈佛大学；6怀斯研究所 波士顿儿童医院干细胞移植计划 项目总结 在过去的十年中，干细胞field有了不朽的发现，证明了 终末分化的细胞，与传统认为的相反，可以恢复到多能性或 直接转化为其他分化的细胞类型。突然之间，再生医学的新方法似乎 触手可及：丢失或损坏的细胞可以由患者特定的fic重新编程的细胞取代，从而提供 需求、兼容、任何所需类型的高质量电池。为了实现这一愿景，Sciencefic++社区做出了 为细胞命运重编程建立健壮和有效的fi协议所做的巨大努力。这些协议是 很大程度上是基于先验的fix(Prefix)合适的转录因子(TF)的异位过度表达，其原理是 这种过度表达可能触发基因调控网络(GRN)状态之间的转换 参与了细胞命运的决定。然而，尽管十年来取得了显著的进步，但这些协议的有效性仍然存在 低，生产的电池的质量往往不能令人满意，许多潜在有用的直接电池命运转化仍然 似乎是不可能的。这些问题对人类诱导的多能干细胞的实际应用构成了巨大的障碍 再生医学中的细胞(HiPSCs)和转分化细胞。 可以说，我们准确和精确地将GRN的TF浓度控制在所需范围内的能力是至关重要的 为了细胞命运重编程的成功。不幸的是，当前基于前fi结合的TF的过度表达的协议 没有表现出这种关键的能力。为了解决这个问题，我们提出了一种全新的细胞命运方法。 本项目中的重新编程：我们用TF的反馈过度表达取代了前fix的过度表达，这是我们 用体内合成的遗传反馈控制器电路实现。在此电路中，过度表达级别不是 PriorifiXx，并根据期望的Tf浓度和实际Tf浓度之间的差异进行调整。因此，它可以 准确和精确地将TFS的浓度控制到所需的值，而不依赖于内源性GRN 对这些TF进行监管。我们的研究计划将重点放在hipsc重新编程上，作为评估fifit的好处的试验台。 我们的方法和第二个关于定向分化成血小板的方法，作为直接临床相关的应用。 具体地说，在目标1中，我们建议系统地研究Prefifi过度表达多能性的影响 用于HiPSC重新编程的TFS。在目标2中，我们建议构造并测试合成遗传反馈控制器 同时实现多个TF的反馈过表达的电路。在AIM 3中，我们将利用 用于人类HiPSC重编程和定向分化的合成遗传反馈控制器电路 变成了血小板。该项目将导致显著更高的重新编程EFfi需求，在电池产品中 与目标细胞类型非常相似，并且在未来，在细胞转换方面，今天似乎不可能。更广泛地说， 我们的合成遗传反馈控制器将使科学家和从业者拥有一种新的工具来精确控制 任何内源GRN，特别是参与细胞命运决定的那些GRN的TFS浓度。 1

Synthetic genetic feedback controller circuits to reprogram cell fate PI: Domitilla Del Vecchio1;4 co-PIs: James J. Collins2;4;5;6, Thorsten Schlaeger7, and Ron Weiss2;3;4 1Department of Mechanical Engineering, MIT; 2Department of Biological Engineering, MIT 3Department of Electrical Engineering and Computer Science, MIT 4Synthetic Biology Center, MIT; 5Broad Institute of MIT & Harvard; 6The Wyss Institute 7 Stem Cell Transplantation Program, Boston Children's Hospital PROJECT SUMMARY The past decade has seen monumental discoveries in the stem cell field, with demonstrations that the fate of a terminally differentiated cell, contrary to what was traditionally believed, could be reverted back to pluripotency or directly converted to other differentiated cell types. All of a sudden, new approaches to regenerative medicine seem within reach: lost or damaged cells could be replaced by patient-specific reprogrammed cells, thus providing on- demand, compatible, high-quality cells of any required type. To meet this vision, the scientific community has made tremendous efforts toward establishing robust and efficient protocols for cell fate reprogramming. These protocols are largely based on a priori fixed (prefixed) ectopic overexpression of suitable transcription factors (TFs), with the rationale that this overexpression could trigger transitions among the states of the gene regulatory networks (GRNs) that take part in cell fate determination. Yet, despite a decade of remarkable progress, the efficiency of these protocols remains low, the quality of produced cells is often unsatisfactory, and many potentially useful direct cell fate conversions still seem impossible. These issues pose a formidable obstacle to the practical use of both human induced pluripotent stem cells (hiPSCs) and transdifferentiated cells in regenerative medicine. Arguably, our ability to accurately and precisely steer the concentrations of GRNs' TFs within desired ranges is critical to the success of cell fate reprogramming. Unfortunately, current protocols based on prefixed TFs' overexpression have not demonstrated this critical ability. To address this problem, we propose a completely new approach to cell fate reprogramming in this project: we replace prefixed overexpression with feedback overexpression of TFs, which we realize with an in vivo synthetic genetic feedback controller circuit. Within this circuit, the overexpression level is not a priori fixed and is adjusted based on the discrepancy between desired and actual TF's concentrations. It therefore can accurately and precisely control TFs' concentrations to desired values, independent of the endogenous GRN that also regulates these TFs. Our research plan focuses first on hiPSC reprogramming as a test-bed for evaluating the benefit of our approach and second on directed differentiation of hiPSCs into platelets as a directly clinically relevant application. Specifically, in AIM 1, we propose to systematically investigate the efficacy of prefixed overexpression of pluripotency TFs for hiPSC reprogramming. In AIM 2, we propose to construct and test the synthetic genetic feedback controller circuits that implement feedback overexpression of a number of TFs concurrently. In AIM 3, we will leverage the synthetic genetic feedback controller circuits for human hiPSC reprogramming and for directed differentiation of hiPSCs into platelets. This project will result in substantially higher reprogramming efficiencies, in cell products that more closely resemble the target cell type, and in the future, in cell conversions that today seem not possible. More broadly, our synthetic genetic feedback controllers will empower scientists and practitioners with a new tool to accurately control the TFs' concentrations of any endogenous GRNs and, in particular, of those GRNs involved in cell fate determination. 1