Developing Novel Chemo-optogenetic Tools for in Vivo Applications

项目摘要/摘要 光遗传学和化学光遗传学是用光调节细胞活动的有力工具。这些工具 通过为询问神经回路功能提供必要的手段来加速神经科学研究。 视网膜疾病的光遗传疗法的临床试验已经在进行中。这些技术的一些局限性 目前的工具包括通常很小的光感电流，它们操纵特定细胞的能力有限 深层组织的活性，对非法生理效应的强劲转基因表达的需要，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-光遗传工具将 解决其中的许多问题。我之前开发了一种基于HIGH的新型化学光遗传工具 适用于调节神经细胞和非神经细胞活动的电导型TRPA1通道 活着。我还开发并执行了一个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质的分子光开关。我是 描述从该屏幕确定的两个命中(目标1，K99阶段)。一种是阶梯功能化疗- 基于TRPA1通道的光发生系统。这一新系统将允许光控通道 开/关，进一步增强了TRPA1实用程序。第二个是基于TRPV1的化学-光遗传系统 频道。我的化学-光遗传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 一种生理上更相关的光诱导动作电位的产生。我也会把斑马鱼 光诱导运动反应筛选法特异性识别内源性蛋白靶向分子 光谱在近红外范围内的光开关(目标3，R00相)。近红外光的使用允许 以便更深入地渗透到组织中，并与现有的光遗传工具和生物传感器成像兼容。 总体而言，我提出的研究将产生新的化学-光遗传工具，并将其改进为Unitary 通道电导，光控制更深层组织的开/关活动，不需要或不需要很低水平的 外源基因表达。我的研究也将为发现新的化学物质创造一个平台 模仿自然细胞活动的光遗传致动器。我开发的下一代工具将增强我们的 能够解剖生物过程，如大脑的复杂神经元网络，并加速 光遗传学的潜在临床应用。我的多样化的导师、顾问和合作者团队 选择既是为了确保我的成功，又是为了在与该项目相关的相关领域进一步培训我 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 Plans将为我提供技术技能和知识，以开发用于In的新型化学-光遗传执行器 活体神经科学应用和更多领域，并为成功过渡到 独立研究人员。

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.