AnteroTag, a Novel Method for Trans-Synaptic Delivery of Active Agents to Map and Modify Anterograde Populations

项目总结 大脑计划的一个目标是开发和验证绘制和操纵神经回路的新工具。这个 行为相关回路的定义和控制需要逆行和顺行跨突触 在体内表现良好的技术。在这个项目中，我们追求的是开发一种小蛋白的长期目标 货物顺行交付的标签(“AnteroTag”)。AnteroTag的新概念是利用 靶向突触小泡的标记有效载荷的神经传递的内源性生物学 囊泡释放、突触后进入和所需动作。我们的调查财团建议利用 以桥小脑回路为模型管道，进一步提炼和严格验证AnteroTag。作为人类的轴突 基底桥神经元(苔藓纤维)在小脑颗粒细胞上形成特定的和定量的突触， 桥小脑回路提供了一个已建立的和保守的模型回路来确定 AnteroTag改变了它的性能。该财团的调查人员将利用他们各自领域的 量化AnteroTag的特异性和生物兼容性的专业知识，以当前的状态为基准 最先进的顺行病毒载体HSV-129；这些在小鼠身上的分析将包括显微镜、电生理学和 活体动物的活体活动成像。一旦管道确定最具体和最安全的版本 在桥小脑回路中，我们将测试当传递给不同的起跑者时的AnteroTag性能 成群的神经元。AnteroTag的广泛用途将通过测试其有效性来确定，以提供 各种遗传编码的标记和修饰，并通过测试AnteroTag衍生品在 进入二级顺行种群。具有安全性、特异性和实用性，从而严格地 在小鼠身上演示，我们将测试在高等脑桥小脑环的表现 包括树鼠、雪貂和NHP在内的有机体。这项提议的结果将是发展， 一种跨突触顺行递送生物的新技术的验证和实现 跨多个大脑区域和多个物种的毒剂。因此，如果成功，我们的项目最终将有助于 开发靶向细胞型和电路特异性疗法来治疗脑部疾病。

PROJECT SUMMARY A goal of the BRAIN initiative is to develop and validate novel tools to map and manipulate neural circuits. The definition and control of behaviorally relevant circuits requires both retrograde and anterograde trans-synaptic technologies that perform well in vivo. In this project, we pursue the long-term goal of developing a small protein tag for the anterograde delivery of cargos (an “AnteroTag”). The novel concept of AnteroTag is to leverage the endogenous biology of neurotransmission to target tagged payloads to synaptic vesicles where they undergo vesicular release, postsynaptic entry, and desired action. Our consortium of investigators proposes to utilize the pontocerebellar circuit as a model pipeline to further refine and rigorously validate AnteroTag. As the axons of basal pontine neurons (mossy fibers) make specific and quantitative synapses upon cerebellar granule cells, the pontocerebellar circuit affords an established and conserved model circuit to determine how iterations of AnteroTag alter its performance. The investigators of the consortium will leverage their respective areas of expertise to quantify the specificity and biocompatibility of AnteroTag, benchmarked against a current state-of- the-art anterograde viral vector HSV-129; these analyses in mice will include microscopy, electrophysiology, and in vivo activity imaging in behaving animals. Once the pipeline determines the most specific and safe version of AnteroTag in the pontocerebellar circuit, we will assay AnteroTag performance when delivered to diverse starter populations of neurons. The broad utility of AnteroTag will then be determined by testing its efficacy to deliver a variety of genetically encoded markers and modifiers, and by testing AnteroTag derivatives for their utility in accessing second-order anterograde populations. With safety, specificity, and utility thus rigorously demonstrated in mice, we will assay the performance of AnteroTag in the pontocerebellar circuit of higher organisms including tree shrews, ferrets, and NHPs. The outcome of this proposal will be the development, validation, and implementation of a novel technology for the trans-synaptic anterograde delivery of biological agents across multiple brain regions and multiple species. Thus, if successful, our project will ultimately aid in the development of targeted cell-type and circuit-specific therapeutics to treat brain disorders.