Molecular Imaging for Detection of Synthetic Biology Circuits, Oscillators and Toggle Switches in Regenerative Medicine

利用治疗性干细胞的力量为发现新的治疗方法带来了巨大的希望， 许多慢性病和未治愈的疾病。尽管很少有里程碑式的研究，但总体成功有限。 世界各地的研究表明，使用这些细胞是安全的，但是，这些细胞必须首先经过工程改造 所以它们可以完成所需的任务，类似于电子元件的设计方式， 执行计算任务。在合成生物学中，就像在电子学中一样，我们可以使用生物 组件来构建一个电路，可以执行一个非常特定的功能。我们可以不用电子零件 使用生物部分或“生物部分”，它们以最外层的特异性相互作用。在这里，我们寻求 使用三个“生物部分”，这将足以控制干细胞的活动和命运在原位。这是极其 重要的是，到目前为止，还没有办法在不侵入的情况下远程控制体内细胞 穿透身体组织 为此，我们将在干细胞内构建一个生物回路。对于“开关”，我们将使用一种新的蛋白质， 由我们实验室发现的一种基因编码这种新的蛋白质可以感知和响应外部的 电磁场，即，可以通过静磁体或电子装置来接通和关断。一旦 激活它导致钙的释放。钙敏感启动子用作“放大器”。第三部分生物 是一个报告基因，我们将用于可视化的活动，并在未来可以取代的 治疗基因 第一个目标是建立一个能在干细胞中表达“装置”所有生物部分的细胞培养体系 细胞特别是在iPSC衍生的神经元和脂肪细胞衍生的干细胞（ADSC）中。在第二个目标中，我们 将测试两种细胞激活模式第一个是铁磁（不要与顺磁混淆） 纳米颗粒（FMNP）。那些FMNP将用与干细胞膜结合的配体功能化。 这将导致基因的持续激活（“关闭”→“打开”）。这与我们需要诱导 细胞分化为特定谱系或甚至逆转多能性。第二种激活模式是 振荡，由电磁铁引起，我们可以在短时间内交替开关。这 可以产生振荡模式，导致代谢物、激素和药物的循环产生。这 例如，对于糖尿病等疾病，它可以取代每日胰岛素注射， 需要控制药物释放的癌症。在第三个目标中，我们将移植生物工程 干细胞在啮齿动物的大脑，并将监测双方的拨动开关和振荡器在体内使用的报告 基因工程用于MRI和PET。因此，这项创新性的研究是一种独特的方法，过渡合成 从微生物到哺乳动物系统的生物学策略，具有明确的临床转化路径。

Harnessing the power of therapeutic stem cells holds great promises for the discovery of new treatments to many chronic and uncured diseases. Despite few milestone studies, the overall success has been limited. Studies around the world showed that it is safe to use such cells, however, those cells must be first engineered so they can preformed the desired task, similar to the way that electronic component are engineered for preforming computational tasks. In synthetic biology, much like in electronics, we can use biological components to build a circuit that can preform a very specific function. Instead of using electronic parts we can used biological parts or “bio-parts” that interact with the outmost specificity one with another. Here we seek to use three “bio-parts” that will be sufficient to control stem cell activity and fate in situ. This is extremely important because to date, there is no way to remote control cells, inside the body, without invasively penetrating the body tissue. To that end, we will construct a biological circuit inside stem cells. For the “switch” we will use a novel protein, encoded by a gene that was discovered in our lab. This novel protein can sense and respond to an external electromagnetic field, i.e., can be switch on and off by a static magnet or an electronic device. Once it is activated it lead to release of calcium. A calcium sensitive promoter is used as an “amplifier”. The third bio-part is a reporter gene that we will use for visualization of the activity and in the future can be replace by a therapeutic gene. In the first aim we will establish a cell culture system that can express all the bio-parts of the “device” in stem cells. Specifically in iPSCs-derived neurons and adipocytes derived stem cells (ADSCs). In the second Aim we will test two modes of cell activation. The first one is with ferromagnetic (not to be confused with paramagnetic) nanoparticles (FMNPs). Those FMNPs will be functionalized with ligands that bind to the stem cell membrane. This will result in continues activation (“off”→”on”) of genes. This is relevant to cases where we need to induce cell differentiation either to specific linage or even to reverse pluripotency. The second mode of activation is oscillations, induced by an electromagnet, that can we alternately switch on and off for brief time periods. This can create an oscillatory pattern that can result in cyclic production of metabolites, hormones and drugs. This is relevant, for example, for disease such as diabetes where it can replace the daily insulin injection or in cancer where are controlled drug release is required. In the third Aim, we will transplant the bioengineered stem cells in the rodent brain and will monitor both the Toggle switch and the oscillator in vivo using a reporter gene engineered for both MRI and PET. Thus, this innovative study is a unique approach to transition synthetic biology strategies from microorganism to mammalian system with clear path for clinical translation.