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 在神经生物学中有着广泛的应用。这部分是因为许多信号受体和途径， 大脑是共享的，限制了狭隘的工程策略的自由度。为了创造更广泛的 选择性细胞调节的工具，我们建议开发定向进化方法， 对大麻素有反应并提供多种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。所提出的方法应该产生非常 高亲和力受体，其受体：配体偶联物具有经验证的特异性。我们的HAVOC将 与目前的DREADD和DART方法形成对比的是，它们允许使用自然效应器， 但浓度要低得多，本质上是在大脑内源性受体的“雷达罩”之下飞行。 特别是，我们将使用HAVOC来检查疼痛的门理论，并因此作为替代品 大麻素的靶向纳米剂量策略模型，以安全地促进镇痛和对抗成瘾。 作为纳米剂量策略开发的起点，我们将重点关注CB 2受体， 在大脑中稀疏表达，但已知其具有抑制多巴胺能神经元的功能。使用我们 一种新的定向进化方法，分区伙伴复制（CPR），我们将首先进化 CB 2的个体变体，其可以与大麻素b-卡隆烯、大麻二酚 (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和它们的进化 受体与分离的神经元，并直接在小鼠疼痛模型（目的2）。虽然前往诊所将 最终需要将新的受体引入患者体内，可能是通过基因疗法，靶向 蛋白质的产生，特别是神经通路，可能提供了少数可行的方法之一，为慢性 治疗疼痛。在未来，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，因此我们认为HAVOC可能作为 可概括的神经技术工具，用于理解和操纵各种神经功能。

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.