Developing novel chemo-optogenetic tools for in vivo applications

开发用于体内应用的新型化学光遗传学工具

• 批准号: 10318223
• 负责人: Pui Ying Lam
• 金额: $ 24.9万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10318223
• 关键词: Action Potentials; Address; Animals; Area; Basic Science; Biochemistry; Biological Assay; Biological Process; Biology; Biosensor; Brain; Cations; Cells; Chemicals; Clinical; Clinical Trials; Complex; Electrophysiology (science); Embryo; Engineering; Ensure; Foundations; Gene Expression; Generations; Goals; Histocompatibility; Human; Image; Ion Channel; Kinetics; Knowledge; Libraries; Ligands; Light; Mentors; Modification; Molecular; Molecular Target; Motion; Neuroglia; Neurons; Neurosciences; Neurosciences Research; Optics; Organ; Penetration; Phase; Physiological; Physiology; Program Development; Property; Proteins; Research; Research Personnel; Retinal Diseases; Safety; Sodium; Sodium Channel; Solubility; Specificity; Structural Biologist; Structure-Activity Relationship; System; TRPA channel; TRPV1 gene; Technical Expertise; Techniques; Tertiary Protein Structure; Tissues; Training; Transgenes; Transmembrane Domain; Whole Organism; Zebrafish; azobenzene; base; clinical application; immunoreaction; in vivo; light scattering; neural circuit; next generation; novel; optogenetics; preservation; protein structure; response; screening; small molecule; success; tool; tool development; transgene expression; ultraviolet; voltage

Project Summary/Abstract Optogenetics and chemo-optogenetics are powerful tools for modulating cell activities with light. These tools accelerate neuroscience research by providing the necessary means for interrogating neural circuit function. Clinical trials of optogenetic therapy for retinal diseases are already underway. Some of the limitations of these current tools include a generally small light-induced current, their limited ability to manipulate specific cell activity in deep tissue, the need for robust transgene expression to illicit physiological effects, and safety concerns over long-term exogenous transgene expression. The novel chemo-optogenetic tools I develop will address many of these issues. I previously developed a novel chemo-optogenetic tool based on the high conductance TRPA1 channel which is suitable for modulating both neuronal and non-neuronal cell activity in vivo. I have also developed and performed a small molecule screen based on the zebrafish light-induced motion response and discovered molecular photoswitches that target endogenous vertebrate proteins. I am characterizing two hits identified from this screen (Aim 1, K99 phase). One is a step-function chemo- optogenetic system based on the TRPA1 channel. This new system will allow for light-controlled channel ON/OFF, further enhancing TRPA1 utility. The second is a chemo-optogenetic system based on the TRPV1 channel. The next phase of my chemo-optogenetic tool-development program is to enhance TRPA1 channel selectivity for sodium while preserving its high channel conductance (Aim 2, K99/R00 phase). This will provide a more physiologically relevant light-induced generation of action potentials. I will also extend the zebrafish light-induced motion response screening assay to specifically identify endogenous protein-targeting molecular photoswitches with spectra in the near infrared range (Aim 3, R00 phase). The use of near infrared light allows for deeper penetration into tissues and for compatibility with existing optogenetic tools and biosensor imaging. Overall, my proposed research will generate novel chemo-optogenetic tools with improvements to unitary channel conductance, light-controlled ON/OFF activity in deeper tissue, and require no or low levels of exogenous gene expression. My research will also create a platform for the discovery of novel chemo- optogenetic actuators that mimic natural cell activity. The next generation tools I develop will enhance our ability to dissect biological processes such as the complex neuronal network of the brain and accelerate the potential clinical use of optogenetics. My diverse team of mentors, advisors and collaborators have been chosen to both ensure my success and to further my training in the relevant areas associated with this project such as ion channel biology, chemical biology, electrophysiology, optogenetics and neuroscience. My training plan will equip me with technical skills and knowledge for developing novel chemo-optogenetic actuators for in vivo neuroscience applications and beyond, and provide a foundation for a successful transition into an independent researcher.

项目概要/摘要 光遗传学和化学光遗传学是用光调节细胞活动的强大工具。这些工具 通过提供询问神经回路功能的必要手段来加速神经科学研究。 光遗传学治疗视网膜疾病的临床试验已经在进行中。这些的一些限制 当前的工具包括通常较小的光感应电流，它们操纵特定细胞的能力有限 深层组织的活性、需要强大的转基因表达来产生非法的生理效应以及安全性 对长期外源转基因表达的担忧。我开发的新型化学光遗传学工具将 解决其中许多问题。我之前开发了一种基于高通量的新型化学光遗传学工具 电导 TRPA1 通道，适用于调节神经元和非神经元细胞的活性 体内。我还开发并进行了基于斑马鱼光诱导的小分子筛选 运动响应并发现了针对内源脊椎动物蛋白的分子光开关。我是 表征从此屏幕中识别出的两次命中（目标 1，K99 阶段）。一种是阶梯功能化学- 基于 TRPA1 通道的光遗传学系统。这个新系统将允许光控通道 ON/OFF，进一步增强 TRPA1 实用性。第二个是基于TRPV1的化学光遗传学系统 渠道。我的化学光遗传学工具开发计划的下一阶段是增强 TRPA1 通道 对钠的选择性，同时保持其高通道电导（目标 2，K99/R00 相）。这将提供 生理上更相关的光诱导动作电位的产生。我也会延长斑马鱼 光诱导运动反应筛选试验，特异性识别内源性蛋白质靶向分子 具有近红外范围光谱（Aim 3，R00 相）的光电开关。使用近红外光可以 更深入地渗透到组织中，并与现有的光遗传学工具和生物传感器成像兼容。 总的来说，我提出的研究将产生新颖的化学光遗传学工具，并改进单一 通道电导，深层组织中的光控开/关活动，并且不需要或需要低水平的 外源基因表达。我的研究还将为发现新化学物质创建一个平台 模仿自然细胞活动的光遗传学执行器。我开发的下一代工具将增强我们的 剖析生物过程（例如大脑复杂的神经元网络）并加速 光遗传学的潜在临床应用。我的导师、顾问和合作者组成的多元化团队 选择我是为了确保我的成功并进一步加强我在与该项目相关领域的培训 例如离子通道生物学、化学生物学、电生理学、光遗传学和神经科学。我的训练 该计划将使我具备开发新型化学光遗传学执行器的技术技能和知识 体内神经科学应用及其他领域，并为成功过渡到 独立研究员。