Design of Fe2+ and H2O2 Induced Proximity Functionalized Imaging Probes for the Control of Cellular Functions

项目摘要 基因/细胞疗法终于成熟了，这要归功于创新和 提高生物技术水平。尽管如此，许多挑战，特别是毒性和不受欢迎/失控 免疫反应仍然存在。这项研究提案解决了这些问题，并代表了关键的第一步 开发新的、更有效的细胞/基因疗法。为此，我们提出了一种新的合成方法 被称为“环境刺激诱导邻近(ESiP)”的时空操纵生物技术 细胞功能。新的策略可以克服已建立的化学诱导邻近的困难。 (CIP)方法调解内源性细胞信号转换为量身定制的细胞功能 时空精确度。为了证明可行性，我们建议开发荧光和 化学发光Fe(II)和H_2O_2响应ESiP化学诱导剂，因为Fe(II)和H_2O_2是重要的 与多种生物功能和疾病相关的信号分子，以及赤霉素(GA) 脱落酸(ABA)是公认的低毒化学诱导剂。具体地说，我们将设计， Fe2+和H_2O_2响应荧光和化学发光ESiP诱导剂的合成、测试和优化 (特定目标1-2)，并在细胞中测试它们，并构建ESiP介导的“与”布尔逻辑门以控制 有条件地生产阿尔茨海默病治疗蛋白(特定目标3)。这些研究将证明，这些“聪明的” 声学探针具有特异性地检测内源性Fe(II)和H_2O_2的能力，并能够触发 原始生物活性化学诱导剂GA和ABA的释放，进而诱导下游生物 功能，如荧光素酶的表达和产生神经元保护性BDNF蛋白。技术 最终将转变为用于疾病治疗的全新细胞/基因疗法，具有显著的 减少副作用。预计这一强大而通用的战略可以与各种 现有的合成生物学分子部件和工具，用于构建新的细胞遗传和信号电路 产生新的功能，作为生物医学研究的工具箱和各种疾病治疗的治疗方法。

Project Summary Gene/cell therapies finally come of age thanks to the fundamental advances made by innovative and improved biotechnologies. Nonetheless, many challenges particularly toxicity and undesired/uncontrolled immune response remain. This research proposal addresses these concerns and represents a critical first step toward developing novel, more effective cell/gene therapy. Toward this end, we propose a novel synthetic biology technology termed “Environment Stimuli-Induced Proximity (ESIP)” to spatiotemporally manipulate cellular functions. The new strategy can overcome the difficulty of the established chemically induced proximity (CIP) method to mediate the translation of endogenous cellular signals into tailored cellular functions with spatiotemporal precision. To demonstrate the feasibility, we propose to develop fluorescent and chemiluminescent Fe(II) and H2O2 responsive ESIP chemical inducers because Fe(II) and H2O2 are important signal molecules, which are associated with numerous biological functions and diseases, and gibberellin (GA) and abscisic acid (ABA) are established chemical inducers with low toxicity. Specifically, we will design, synthesize, test and optimize Fe2+ and H2O2-responsive fluorescent and chemiluminescent ESIP inducers (Specific Aims 1-2) and test them in cells and construct ESIP-mediated “AND” Boolean logic gates to control the conditional production of AD therapeutic proteins (Specific Aim 3). These studies will prove that these `smart' theranostic probes possess the ability to sense endogenous Fe(II) and H2O2 specifically, and the ability to trigger the release of original bioactive chemical inducers GA and ABA, which then induce downstream biological functions such as luciferase expression and the production of neuron protective BDNF proteins. The technology will ultimately be transformed into entirely new cell/gene therapies for disease treatment with significantly reduced side effects. It is expected that this powerful and general strategy can be integrated with a variety of existing synthetic biology molecular parts and tools to build new cellular genetic and signaling circuitries to generate new functions as toolbox for biomedical research and as therapeutics for various disease treatment.