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: 10556337
• 负责人: John Douglas Hulleman
• 金额: $ 7.81万
• 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 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10556337
• 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; biological adaptation to stress; cell type; constitutive expression; 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.

项目摘要 在空间和时间上控制细胞信号传导的能力是生物体的基本要求， 组织发育/功能。作为一种器官，眼睛也不能免除这些要求。事实上，可以说， 视网膜甚至比其他组织对环境或遗传干扰更敏感， 将光转换为最终被大脑识别的电化学电势。因此，不受监管 基因表达（也称为表型毒性）在不适当的时间（时间不可知），或泛视网膜 使用小分子激活/抑制细胞信号传导，而不管细胞类型（空间不可知）， 潜在的危害生理信号与可怕的后果。为了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利用了一个 化学生物学方法涉及小鼠视网膜中的去稳定化结构域（DDs）。这些DD工具允许 通过加入外源性小分子稳定剂对蛋白质丰度进行条件控制 （对于E.大肠杆菌二氢叶酸还原酶[DHFR]结构域），并可以实现时间 控制视网膜内接近生理相关的时间尺度。我们设想，这些方法将 提供理想化的治疗剂，其可以靶向受疾病影响的所需细胞类型，并且根据定义的 的时间表.该R21的总体目标是大幅扩展 可用于体内条件性调节视网膜应激反应的可用小分子探针 使用DD进行信令。这项工作很重要，因为它将使科学界能够 以越来越高的时空精度和灵活性询问视网膜生物现象，它将测试 有条件基因疗法的意识形态界限和治疗效用。