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.

项目摘要/摘要 光遗传学和化学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。 视网膜疾病的光遗传学疗法的临床试验已经在进行中。其中的一些局限性 当前工具包括通常小的光引起的电流，它们操纵特定电池的能力有限 在深层组织中的活性，需要强大的转基因表达对非法生理影响和安全性的需求 担心长期外源转基因表达。我开发的新型化学典型发育工具将 解决其中许多问题。我以前基于高 电导TRPA1通道，适用于调节神经元和非神经元细胞活性 体内。我还根据斑马鱼诱导的 运动响应并发现了靶向内源性脊椎动物蛋白的分子照片开关。我是 表征从此屏幕上标识的两个命中（AIM 1，K99相）。一个是阶跃功能化学疗法 基于TRPA1通道的光遗传系统。这个新系统将允许光制频道 开/关，进一步增强TRPA1实用程序。第二个是基于TRPV1的化学灭节系统 渠道。我的化学遗传学工具开发计划的下一阶段是增强TRPA1通道 钠的选择性，同时保留其高通道电导率（AIM 2，K99/R00相）。这将提供 具有更相关的光引起的动作电位的更相关的。我还将扩展斑马鱼 光诱导的运动响应筛选测定法，以特异性识别内源性蛋白质靶向分子 带有光谱的照片开关在近红外范围内（AIM 3，R00阶段）。近红外光的使用允许 为了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。 总体而言，我提出的研究将生成新颖的化学植物学工具，并改善统一 通道电导，在较深的组织中进行轻度控制/关闭活动，不需要或低水平 外源基因表达。我的研究还将创建一个平台，以发现新型化学疗法 模仿天然细胞活性的光遗传驱动器。我开发的下一代工具将增强我们的 能够解剖生物学过程，例如大脑的复杂神经元网络并加速 光遗传学的潜在临床使用。我多样的导师，顾问和合作者团队已经 选择既要确保我的成功，又要进一步在与该项目相关的相关领域进行培训 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 计划将使我具备技术技能和知识，以开发新型的化学源性执行器 体内神经科学的应用及其他，并为成功过渡到一个 独立研究员。