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

项目总结 细胞分化是由整合多种感觉输入的表观遗传调控系统控制的。 是时候指导长期细胞命运的决定了。在合成基因设计中利用表观遗传调控 电路将极大地增强合成生物学。基于染色质控制的合成表观遗传回路 与当前形式人工细胞存储相比，状态呈现许多吸引人的优点(例如，重组酶或 基于Cas9的开关/级联)，这可能在可扩展性、稳定性和时间控制方面受到限制。天然 表观遗传系统在不改变遗传信息的情况下支持稳定的记忆状态，可以诱导状态变化 以确定性或随机性的方式，并且仍然保持可逆性。 我们建议生成一个合成工具箱来响应于用户指定的输入来调节染色质状态， 从而允许构建具有关键表观遗传属性的电路，例如记忆、命运分叉和 时间控制的基因表达。我们建议的电路将设计为a)响应各种 通过合成Notch受体的细胞外输入信号，b)根据持续时间区分输入信号，以特定 仅在对持续环境刺激的反应中才能诱导细胞命运改变，c)时间控制基因 促进细胞行为序列的表达程序，最终d)建立分歧 分化状态，以允许在细胞群体内进行功能上有利的专门化。我们的工作将 将在CAR T细胞免疫治疗的试验床上开发，这是哺乳动物细胞的主要应用领域 工程学可以从结合了表观遗传记忆和时间的合成电路中受益匪浅 控制能力。然而，由此产生的表观遗传工具包和电路将适用于更大的 工程细胞的范围，包括在再生医学和更广泛的细胞疗法中的应用。

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.