Synthetic biology for the chemogenetic manipulation of pain pathways

用于疼痛通路化学遗传学操纵的合成生物学

• 批准号: 10017883
• 负责人: Andrew D Ellington
• 金额: $ 23.16万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017883
• 关键词: Absence of pain sensation; Affinity; Analgesics; Animal Model; Beta-caryophyllene; Biological Assay; Biological Sciences; Biology; Brain; CNR2 gene; Cannabidiol; Cannabinoids; Cells; Chronic; Clinic; Collaborations; Complex; Coupled; Couples; Cultured Cells; Development; Directed Molecular Evolution; Discipline; Dose; Electrophysiology (science); Emulsions; Engineering; Enzymes; Flying body movement; Future; G-Protein-Coupled Receptors; Genes; Individual; Ion Channel; Lead; Learning; Libraries; Ligands; Memory; Methods; Minor; Modeling; Movement; Mus; Neural Pathways; Neurobiology; Neurons; Neurophysiology - biologic function; Oils; Pain; Pain management; Pathway interactions; Patients; Performance; Pheromone; Polymerase; Prevention; Production; Proteins; Radar; Receptor Signaling; Scheme; Sensory Receptors; Signal Pathway; Signal Transduction; Testing; Variant; Water; Work; Yeasts; addiction; cannabinoid drug; combat; designer receptors exclusively activated by designer drugs; dopaminergic neuron; gene therapy; genetic regulatory protein; mouse model; nano; nanomolar; novel; pain model; pain relief; receptor; relating to nervous system; selective expression; side effect; synthetic biology; theories; tool

Project Summary The methods of synthetic biology have transformed practice throughout the biological sciences, but have yet to find wide application in neurobiology. This is in part because many signaling receptors and pathways in the brains are shared, limiting the latitude for narrowly targeted engineering strategies. To create a wider range of tools for selective cell modulation, we propose to develop directed evolution methods that will generate orthogonal neural receptors that respond to cannabinoids and offer multiple different chemogenetic control points across the brain and thereby open the way to a synthetic neurobiology. The proposed methods should yield very High Affinity receptors, that have Validated Orthogonalities for their receptor:ligand Couples. Our HAVOCs will stand in contrast to current DREADD and DART approaches in that they will allow the use of natural effectors, but at much lower concentrations, in essence flying below the ‘radar cover’ of endogenous receptors in the brain. In particular, we will use HAVOCs to examine the gate theory of pain, and in consequence serve as a surrogate model for targeted nano-dosing strategies for cannabinoids to safely promote analgesia and combat addiction. As a starting point for the development of nano-dosing strategies, we will focus on the CB2 receptor, which is sparsely expressed in the brain, but which has known functions in inhibiting dopaminergic neurons. Using our novel directed evolution method, Compartmentalized Partnered Replication (CPR), we will initially evolve individual variants of CB2 that can interact with high affinity with the cannabinoids b-caryophyllene, cannabidiol (CBD), and other minor cannabinoids (Aim 1). We will proof the utility of these compounds and their evolved receptors with isolated neurons and directly in a mouse model for pain (Aim 2). While movement to the clinic will ultimately require introduction of novel receptors into patients, likely via gene therapies, the ability to target protein production in particular neural pathways may provide one of the few viable methods for the chronic treatment of pain. Into the future, the directed evolution strategies we have developed are fungible between multiple different receptors and receptor types, and we suggest that HAVOCs may therefore serve as generalizable neurotechnological tools to understand and manipulate a variety of neural functions.

项目摘要 合成生物学的方法在整个生物学科学中都改变了实践，但尚未 在神经生物学中找到广泛的应用。这部分是因为许多信号接收器和路径 大脑是共享的，限制了针对目标的工程策略的纬度。创建更广泛的范围 选择性单元调制的工具，我们建议开发将生成的定向演化方法 对大麻素反应并提供多种不同的化学遗传控制点的正交神经接收器 在整个大脑中，从而为合成神经生物学开辟了道路。提出的方法应产生非常 高亲和力受体已经验​​证了其受体的正交性：配体夫妇。我们的破坏会 与当前的Dreadd和Dart方法相反，它们将允许使用自然效应， 但是，在较低的浓度下，本质上飞到大脑内源性受体的“雷达覆盖率”以下。 特别是，我们将使用破坏来检查疼痛的门理论，因此作为替代物 针对大麻素的靶向纳米剂量策略的模型，以安全地促进镇痛和作战成瘾。 作为开发纳米剂量策略的起点，我们将专注于CB2受体，这是 在大脑中稀疏表达，但在抑制多巴胺能神经元中已知功能。使用我们的 新颖的定向进化方法，分室化的合作复制（CPR），我们最初将进化 CB2的个体变体可以与大麻素B-核酚，大麻二醇相互作用 （CBD）和其他次要大麻素（AIM 1）。我们将证明这些化合物的实用性及其进化 具有孤立神经元的受体，直接在小鼠模型中进行疼痛（AIM 2）。而诊所的运动将 最终需要将新型受体引入患者，可能是通过基因疗法，靶向靶向的能力 蛋白质产生特别是神经途径可能是慢性的少数可行方法之一 治疗疼痛。在未来，我们制定的定向演化策略在 多种不同的受体和受体类型，我们建议破坏可以作为 可概括的神经技术工具，以理解和操纵各种神经功能。