Synthetic Genetic Controller Circuits to Reprogram Cell Fate

重新编程细胞命运的合成遗传控制器电路

基本信息

批准号：
9367460
负责人：
JAMES J COLLINS
金额：
$ 63.2万
依托单位：
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-09-01 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9367460
关键词：
Address Antibodies BCL1 Oncogene BMI1 gene Back Beds Benchmarking Biological Biology Blood Cells Blood Platelets Boston CD34 gene Cell Differentiation process Cells Communities Data Dose Electrical Engineering Engineering Epigenetic Process Feedback Fibroblasts Fluorescence Future Genetic Goals Hematopoietic stem cells Human Institutes Knowledge Label Lead Measures Mechanics Methods MicroRNAs Modality Patients Pediatric Hospitals Platelet Transfusion Process Protocols documentation RNA Regenerative Medicine Regulator Genes Research Scientist Stem cell transplant Stem cells Study models System Techniques Testing Transcriptional Regulation Transfection Umbilical Cord Blood Vision base c-myc Genes cell injury cell type clinical translation clinically relevant computer science design feeding fetal improved in vivo induced pluripotent stem cell insight mathematical analysis mathematical model novel novel strategies overexpression pluripotency predictive modeling prevent programs promoter response success tool transcription factor transcriptome sequencing

项目摘要

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

合成遗传反馈控制器电路重新编程细胞命运 PI：多米蒂拉·德尔·维奇奥1;4 联合 PI：James J. Collins2;4;5;6、Thorsten Schlaeger7 和 Ron Weiss2;3;4 1麻省理工学院机械工程系；2麻省理工学院生物工程系 3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&哈佛大学布罗德研究所；6威斯研究所 7 波士顿儿童医院干细胞移植项目项目概要过去十年，干细胞领域取得了巨大的发现，证明了干细胞的命运与传统观点相反，终末分化细胞可以恢复多能性或突然之间，再生医学的新方法似乎出现了。触手可及：丢失或损坏的细胞可以被患者特异性重编程细胞取代，从而提供为了实现这一愿景，科学界提出了任何所需类型的、兼容的、高质量的细胞。这些方案对于建立强有力的、有效的细胞命运重编程努力是巨大的。很大程度上基于合适转录因子（TF）的先验固定（前缀）异位过度表达，其基本原理这种过度表达可能会触发基因调控网络（GRN）状态之间的转变，然而，尽管十年来取得了显着进展，这些方案的效率仍然存在。低，产生的细胞质量往往不能令人满意，许多潜在有用的直接细胞命运转换仍然存在这些问题似乎对人类诱导多能干细胞的实际应用构成了巨大的障碍。细胞（hiPSC）和再生医学中的转分化细胞。可以说，我们准确、精确地将 GRN 的 TF 浓度控制在所需范围内的能力至关重要不幸的是，目前基于前缀 TF 过度表达的方案已经无法实现细胞命运重编程的成功。为了解决这个问题，我们提出了一种全新的细胞命运方法。在这个项目中重新编程：我们用 TF 的反馈过度表达替换前缀过度表达，我们通过体内合成遗传反馈控制器电路实现。在该电路中，过度表达水平不是一个。先验固定并根据所需 TF 浓度与实际 TF 浓度之间的差异进行调整。准确、精确地控制 TF 的浓度至所需值，独立于内源 GRN 我们的研究计划侧重于 hiPSC 重编程作为评估其益处的测试平台。我们的方法和第二个方法是将 hiPSC 定向分化为血小板，作为直接的临床相关应用。具体来说，在 AIM 1 中，我们建议系统地研究多能性前缀过度表达的功效用于 hiPSC 重编程的 TF 在 AIM 2 中，我们建议构建并测试合成遗传反馈控制器。同时实现多个 TF 的反馈过度表达的电路在 AIM 3 中，我们将利用用于人类 hiPSC 重编程和 hiPSC 定向分化的合成遗传反馈控制器电路该项目将大大提高细胞产品的重编程效率。与目标细胞类型非常相似，并且在未来，在今天看来不可能的细胞转换中，我们的合成遗传反馈控制器将为科学家和从业者提供一种新工具来准确控制任何内源 GRN 的 TF 浓度，特别是那些参与细胞命运决定的 GRN 的浓度。 1