Synthetic biology for the chemogenetic manipulation of pain pathways

项目摘要 合成生物学的方法已经改变了整个生物科学的实践，但还没有。 在神经生物学中有广泛的应用。这在一定程度上是因为体内许多信号受体和通路 大脑是共享的，这限制了狭隘目标工程战略的自由度。创建更广泛的 用于选择性细胞调制的工具，我们建议开发定向进化方法，以产生 对大麻素做出反应并提供多个不同化学发生控制点的正交神经受体 从而为合成神经生物学开辟了道路。建议的方法应该会产生非常大的收益 高亲和力受体，已证实其受体的正交性：配基对。我们的哈沃克人会 与当前的DREADD和DART方法形成对比，因为它们将允许使用自然效应器， 但在低得多的浓度下，实质上是飞行在大脑内源性受体的“雷达掩护”之下。 特别是，我们将使用Havocs来检验痛苦的门理论，并因此充当代理 针对大麻类药物的定向纳米剂量战略的模型，以安全地促进止痛和打击成瘾。 作为发展纳米剂量策略的起点，我们将重点研究CB2受体，它是 在大脑中少量表达，但已知具有抑制多巴胺能神经元的功能。使用我们的 新的定向进化方法，分区伙伴复制(CPR)，我们将进行初步进化 CB2的个别变异体可以与大麻类化合物b-石竹烯、大麻二酚高亲和力相互作用 (CBD)和其他次要的大麻素(目标1)。我们将证明这些化合物及其进化的效用 受体与分离的神经元结合，并直接在小鼠疼痛模型中发挥作用(目标2)。而搬到诊所将会 最终需要将新的受体引入患者体内，可能是通过基因治疗，靶向的能力 蛋白质的产生，特别是神经通路，可能是治疗慢性精神分裂症的少数可行方法之一。 疼痛的治疗。展望未来，我们开发的定向进化策略可以在 多种不同的受体和受体类型，我们认为Havocs可能因此作为 可概括的神经技术工具，用于理解和操作各种神经功能。

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.