Synthetic epigenetic circuits: tunable cell fate switches controlled by dynamic and combinatorial inputs

项目摘要 细胞分化是由表观遗传调控系统控制的，该系统整合了多种感觉输入， 决定细胞长期命运的时间。在合成基因设计中利用表观遗传调控 电路将大大增强合成生物学。基于控制染色质的合成表观遗传回路 目前的人工细胞记忆形式（例如重组酶或 基于Cas9的交换机/级联），这可能在可扩展性、稳定性和时间控制方面受到限制。自然 表观遗传系统支持稳定的记忆状态，而不改变遗传信息，可以诱导状态变化， 以确定性或随机方式，且仍然保持可逆性。 我们建议生成一个合成工具箱来调节染色质状态，以响应用户指定的输入， 从而允许构建具有关键表观遗传特性的电路，例如记忆，命运分叉和 时间控制的基因表达。我们提出的电路将被设计为a）响应各种 通过合成Notch受体的细胞外输入线索，B）通过持续时间区分输入，以特异性地 仅响应持续环境刺激诱导细胞命运改变，c）时间控制基因 表达程序，以促进细胞行为的序列，最后d）建立不同的 分化状态，以允许在细胞群体内功能上有利的特化。我们的工作将 将在CAR T细胞免疫疗法的试验平台上开发，这是哺乳动物细胞免疫疗法的主要应用领域。 工程，可以大大受益于合成电路，纳入表观遗传记忆和时间 控制能力。由此产生的表观遗传工具包和电路，然而，将适用于一个更大的 一系列工程细胞，包括在再生医学和细胞疗法更广泛的应用。

PROJECT SUMMARY Cellular differentiation is controlled by epigenetic regulatory systems that integrate multiple sensory inputs over time to direct long-term cell fate decisions. Harnessing epigenetic regulation in the design of synthetic gene circuits would greatly augment synthetic biology. Synthetic epigenetic circuits based on controlling chromatin state present many attractive advantages over current forms of artificial cellular memory (e.g. recombinase or Cas9-based switches/cascades) which can be limited in scalability, stability, and temporal control. Natural epigenetic systems support stable memory states without altering genetic information, can induce state changes in either deterministic or stochastic fashion, and still maintain reversibility. We propose to generate a synthetic toolbox to regulate chromatin state in response to user-specified inputs, thereby allowing construction of circuits with key epigenetic properties, such as memory, fate bifurcation and temporally controlled gene expression. Our proposed circuits will be designed to a) respond to a variety of extracellular input cues through synthetic Notch receptors, b) discriminate inputs by duration to specifically induce cell fate changes only in response to persistent environmental stimuli, c) temporally control gene expression programs to promote sequences of cellular behaviors, and finally d) establish divergent differentiation states to allow functionally advantageous specialization within a cell population. Our work will be developed in the testbed of CAR T cell immunotherapy, a major application area for mammalian cellular engineering that could greatly benefit from synthetic circuits that incorporate epigenetic memory and temporal control capabilities. The resulting epigenetic toolkit and circuits, however, will be applicable to a much larger range of engineered cells, including applications in regenerative medicine and cell therapies more broadly.