Engineering human skin fibroblasts with an orthogonal transcriptional circuit conferring glucose-responsive insulin release

在这个项目中，我们将采用一种新颖的方法-利用合成生物学的最新工具来创建合成转录调控子和启动子，以构建一个正交转录回路，使葡萄糖传感和控制相应水平的胰岛素释放。这样的电路将被设计成人类真皮成纤维细胞，这是一种易于基因组工程的自体细胞类型。在可编程转录因子、原代人类细胞培养和合成调节电路/启动子方面的专业知识将支持该项目，并使我们能够定义有效的葡萄糖传感和胰岛素释放的最佳电路设计，并具有适当的反馈电路。我们将利用CRISPR/Cas系统和基于tale的转录激活因子和抑制因子来创建新的合成转录因子，这些转录因子将用于构建合成电路。我们的葡萄糖反应回路的发展将提供一种以高度控制的方式产生胰岛素的方法。这种电路将在人类成纤维细胞中进行测试，成纤维细胞是一种容易获得的人类细胞类型，可以在培养中长期扩增，并且可以很容易地进行基因操作。最后，一旦该电路在体外的葡萄糖感应和胰岛素产生方面得到验证，我们将在免疫功能低下的小鼠皮下移植后测试其在体内的工作效果。从长远来看，设计患者来源的成纤维细胞来产生胰岛素，将允许细胞移植回患者体内，而无需使用刺激性免疫抑制药物或封装设备。

In this project we will take a novel approach - exploiting the latest tools of synthetic biology to create synthetic transcriptional regulators and promoters to construct an orthogonal transcriptional circuit enabling glucose sensing and controlled release of corresponding levels of insulin. Such a circuit will be engineered into human dermal fibroblasts, which are an accessible autologous cell type amenable to genome engineering. Expertise in programmable transcription factors, primary human cell culture and synthetic regulatory circuits/promoters will underpin this project and enable us to define the optimal circuit design for effective glucose sensing and insulin release, with appropriate feedback circuits. We will exploit the CRISPR/Cas system and TALE-based transcriptional activators and repressors to create novel synthetic transcription factors and these will be used to construct the synthetic circuit. The development of our glucose-responsive circuit will provide a means of producing insulin in a highly controlled manner. The circuits will be tested in human fibroblasts, an easily-obtainable human cell type that may be expanded long term in culture and can be readily genetically manipulated. Finally, once the circuit has been validated in vitro with regards to glucose sensing and insulin production, we will test how well this works in vivo following subcutaneous transplantation into immunocompromised mice. In the longer term, engineering patient-derived fibroblasts to produce insulin will allow cells to be grafted back onto the patient without the use of harsh immunosuppressive drugs or encapsulation devices.