Development and application of chemical timers for precise control of protein abundance in the eye

项目摘要 在空间和时间上控制细胞信号的能力是生物体和 组织发育/功能。作为一个器官，眼睛也不能免除这些要求。事实上，可以说， 视网膜比其他组织对环境或遗传干扰更敏感，这些干扰可能会影响 将光转化为最终被大脑识别的电化学势。因此，不受监管 基因表达(也称为表观毒性)在不适当的时间(暂时不可知)，或泛视网膜 使用小分子激活/抑制细胞信号而不考虑细胞类型(空间上不可知)，有 有可能损害生理信号，带来可怕的后果。来规避这些潜在的不利因素 结果，并开发一个理想的基因治疗平台，我们已经彻底验证和使用了一种 涉及小鼠视网膜中不稳定结构域(DDS)的化学生物学方法。这些DD工具允许 通过添加外源添加的小分子稳定剂来条件控制蛋白质丰度 (通常是甲氧苄氨嘧啶，TMP，针对大肠杆菌二氢叶酸还原酶[DHFR]结构域)，并可以实现 控制视网膜内近乎生理相关的时间尺度。我们设想，这种方法将 提供理想的治疗方法，可以针对疾病中受影响的所需细胞类型，并根据定义 时间框架。此R21的总体目标是大幅扩展的新颖性、功能和适用性 可用于体内有条件调节视网膜应激反应的小分子探针 使用DDS发送信号。这项工作很重要，因为它将显著地使科学界能够 随着时空精确度和灵活性的提高，询问视网膜生物现象将测试 条件性基因疗法的思想界限和治疗效用。

Project Summary The ability to spatially and temporally control cellular signaling is a fundamental requirement of organismal and tissue development/function. As an organ, the eye is not exempt from these requirements. In fact, arguably, the retina is even more sensitive than other tissues to environmental or genetic disturbances which can affect the transduction of light into electrochemical potential that is ultimately recognized by the brain. Thus, unregulated gene expression (also called phenotoxicity) during inappropriate times (temporally agnostic), or pan-retina activation/repression of cellular signaling using small molecules regardless of cell type (spatially agnostic), have the potential to compromise physiologic signaling with dire consequences. To circumvent these potential adverse consequences, and to develop an idealized gene-therapy platform, we have thoroughly validated and utilized a chemical biology approach involving destabilizing domains (DDs) in the mouse retina. These DD tools allow for conditional control of protein abundance through the addition of an exogenously-added small molecule stabilizer (typically trimethoprim, TMP, for the E. coli dihydrofolate reductase [DHFR] domain) and can achieve temporal control on near physiologically-relevant timescales within the retina. We envision that such approaches will provide idealized therapeutics that can target desired cell types affected in disease and according to defined timeframes. The overall goal of this R21 is to substantially expand the novelty, capability, and applicability of available small molecule probes that can be used in vivo for conditionally regulating retinal stress responsive signaling using DDs. This work is important because it will significantly enable the scientific community to interrogate retinal biologic phenomena with increasing spatio-temporal precision and flexibility, and it will test the ideological boundaries and therapeutic utility of conditional gene therapies.