Bacteriophytochrome-based optogenetic tools for mammalian gene regulation

描述(申请人提供)：一旦基因工程细胞，如用于修复受损组织的干细胞，用于识别和摧毁肿瘤的免疫细胞，或用于内分泌疾病的激素产生细胞，被输送到哺乳动物宿主中，它们就变得难以控制。这种情况带来了前所未有的风险，这与工程细胞易发生转化和/或故障有关。药物无法区分体内功能正常和功能不良的细胞，而基因内置的安全机制可能被证明是不够的。控制生物过程的光遗传方法提供了化学物质无法比拟的时空分辨率，但紫外线-可见光无法到达深层哺乳动物组织。相比之下，近红外窗口中的光被认为是安全的，可以穿透哺乳动物组织几厘米深，至少比紫外线-可见光深几倍。因此，从外部放置的激光或插入体腔的光导发出的光可以到达内脏，控制生物活动。细菌藻色素是唯一一类能够感知近红外光的感光蛋白。细菌植物色素自动催化结合它们的发色团(胆绿素)，这是天然的 在哺乳动物细胞中制造。这种幸运的情况消除了对外源发色团供应的需要。在这个项目中，我们打算设计基于细菌光敏色素的遗传模块，用于哺乳动物，包括人类的正交基因调控。我们计划为几种小鼠组织优化这些光激活模块。这里开发的光遗传学工具将允许研究人员在活体动物的特定组织中执行有条件的和可逆的基因敲除(或基因激活)，这将加深我们对各种疾病进展的理解，提高我们对哺乳动物发育的知识，并提供对宿主-病原体相互作用的实时洞察。这些工具还将使基因和工程细胞疗法更安全、更智能。

DESCRIPTION (provided by applicant): Once genetically engineered cells, such as stem cells designed to repair damaged tissues, immune cells programmed to recognize and destroy tumors, or hormone-producing cells for endocrine disorders, are delivered into a mammalian host, they become poorly controllable. This situation poses unprecedented risks associated with the predisposition of the engineered cells to transformation and/or malfunction. Drugs cannot distinguish between properly functioning and malfunctioning cells inside the body, while genetically build-in safety mechanisms may prove insufficient. Optogenetic approaches to control biological processes offer spatiotemporal resolution unmatched by chemicals, yet UV-visible light cannot reach deep mammalian tissues. In contrast, light in the near-infrared window is known to be safe and penetrate mammalian tissues to the depth of several centimeters, at least several-fold deeper than UV-visible light. Therefore, light from externally placed lasers or light guides inserted into body cavities can reach internal organs and control biological activitie. Bacteriophytochromes are the only class of photoreceptor proteins that sense near-infrared light. Bacteriophytochromes autocatalytically bind their chromophore (biliverdin) that is naturally made in mammalian cells. This fortunate circumstance obviates the need for exogenous chromophore supply. In this project we intend to engineer bacteriophytochrome-based genetic modules for orthogonal gene regulation in mammals, including humans. We plan to optimize these light-activated modules for several mouse tissues. The optogenetic tools developed here will allow researchers to execute conditional and reversible gene knockouts (or gene activation) in specific tissues of live animals, which will deepened our understanding of progression of various diseases, improve our knowledge of mammalian development, and offer real-time insights into host- pathogen interactions. These tools will also make gene and engineered cell therapies safer and smarter.