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。尽管几乎没有里程碑式的研究，但总体成功是有限的。 世界各地的研究表明，使用这种细胞是安全的，然而，必须首先对这些细胞进行工程改造 这样他们就可以执行所需的任务，类似于设计电子部件的方式 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 组件来构建一种可以预制非常特定功能的电路。代替使用电子部件，我们可以 使用生物部分或“生物部分”，它们相互作用于最外在的特性。在这里，我们试图 使用三个足以在原位控制干细胞活性和命运的“生物部分”。这是极其重要的 重要的是，到目前为止，没有一种方法可以远程控制体内的细胞，而不是无创地 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，即可以通过静电磁铁或电子设备来开启和关闭。一旦它成为现实 激活它会导致钙的释放。一种钙敏感的启动子被用作“放大器”。第三部分--传记 是我们将用于活动可视化的报告基因，并且在将来可以被替换为 治疗性基因。 在第一个目标中，我们将建立一个细胞培养系统，它可以在干细胞中表达“装置”的所有生物部分。 细胞。尤其是IPSCs来源的神经元和脂肪细胞来源的干细胞(ADSCs)。在第二个目标中，我们 将测试两种细胞激活模式。第一种是铁磁性(不要与顺磁性混淆) 纳米颗粒(FMNPs)。这些FMNPs将通过与干细胞膜结合的配体来功能化。 这将导致基因的持续激活(“OFF”→“ON”)。这与我们需要诱导的情况有关 细胞分化到特定的谱系，甚至逆转多能性。第二种激活模式是 由电磁铁引起的振荡，我们可以在短时间内交替开启和关闭。这 可以产生一种振荡模式，从而导致代谢物、激素和药物的循环生产。这 例如，与糖尿病等疾病相关，在这些疾病中，它可以取代日常的胰岛素注射或在 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化的 干细胞在啮齿类动物的大脑中，并将使用报告监测体内的拨动开关和振荡器 基因工程可用于核磁共振和正电子发射计算机断层扫描。因此，这项创新性的研究是一种独特的过渡综合方法 从微生物到哺乳动物系统的生物学策略，为临床翻译提供了明确的路径。

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.