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

精确控制眼内蛋白质丰度的化学计时器的开发和应用

• 批准号: 10354805
• 负责人: John Douglas Hulleman
• 金额: $ 24.6万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10354805
• 关键词: Affect; Affinity; Antioxidants; Binding; Biological; Biological Phenomena; Biology; Brain; Chemicals; Communities; Development; Dihydrofolate Reductase; Disease; Escherichia coli; Eye; Eye diseases; Gene Expression; Genes; Genetic; Glaucoma; Goals; In Vitro; Inherited; Intervention; Light; Lipid Peroxidation; Methods; Mus; Nonexudative age-related macular degeneration; Organ; Outcome; Oxidative Stress; Pathway interactions; Physiologic pulse; Physiological; Positioning Attribute; Proteins; Regulation; Repression; Research Personnel; Retina; Retinal Degeneration; Retinitis Pigmentosa; Series; Signal Pathway; Signal Transduction; Specific qualifier value; Stress; Structure of retinal pigment epithelium; Superoxides; System; Tertiary Protein Structure; Testing; Therapeutic; Time; Tissues; Trimethoprim; Work; achromatopsia; adverse outcome; age related; analog; base; biological adaptation to stress; cell type; flexibility; gene therapy; in vivo; next generation; prevent; promoter; protective effect; proteostasis; response; small molecule; spatiotemporal; tool; transcription factor

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.

项目概要 空间和时间控制细胞信号传导的能力是有机体和生命体的基本要求。 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加敏感，这些干扰可能会影响 将光转换成最终被大脑识别的电化学势。因此，不受监管 不适当时间（时间不可知）或全视网膜的基因表达（也称为表毒性） 使用小分子激活/抑制细胞信号传导，无论细胞类型如何（空间不可知）， 可能会损害生理信号并带来可怕的后果。为了规避这些潜在的不利因素 后果，并开发一个理想化的基因治疗平台，我们已经彻底验证并利用了 涉及小鼠视网膜不稳定域（DD）的化学生物学方法。这些 DD 工具允许 通过添加外源小分子稳定剂有条件控制蛋白质丰度 （通常是甲氧苄啶，TMP，用于大肠杆菌二氢叶酸还原酶 [DHFR] 结构域）并且可以实现时间 控制视网膜内接近生理相关的时间尺度。我们预计此类方法将 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 时间范围。 R21 的总体目标是大幅扩展 R21 的新颖性、功能和适用性 可用于体内条件性调节视网膜应激反应的小分子探针 使用 DD 发送信号。这项工作很重要，因为它将极大地帮助科学界 以不断提高的时空精度和灵活性来询问视网膜生物现象，并将测试 条件基因疗法的意识形态界限和治疗效用。