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

项目摘要 BRAIN计划的一个目标是开发和验证新的工具来映射和操纵神经回路。的 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，我们追求的长期目标是开发一种小蛋白质， 用于货物的顺行递送的标签（“AnteroTag”）。AnteroTag的新概念是利用 神经传递的内源性生物学将标记的有效载荷靶向突触囊泡， 囊泡释放、突触后进入和期望的作用。我们的研究人员联盟建议利用 桥小脑回路作为模型管道，以进一步完善和严格验证AnteroTag。当神经元的轴突 基底脑桥神经元（苔藓纤维）在小脑颗粒细胞上形成特异性和定量的突触， 脑桥小脑回路提供了一个建立和保存的模型电路，以确定如何迭代的 AnteroTag改变其性能。联合体的调查人员将利用各自的领域， 专业知识来量化AnteroTag的特异性和生物相容性，以当前的 现有技术的顺行病毒载体HSV-129;这些小鼠分析将包括显微镜检查、电生理学和 in vivo体内activity活动imaging成像in behaving表现animals动物.一旦管道确定了最具体、最安全的 AnteroTag在脑桥小脑回路中的作用，我们将分析AnteroTag在传递给不同启动器时的性能 神经元群体。然后将通过测试其有效性来确定AnteroTag的广泛效用， 各种遗传编码的标记和修饰物，并通过测试AnteroTag衍生物在 访问二级顺行种群。安全性、特异性和实用性， 在小鼠中证明，我们将测定AnteroTag在较高的脑桥小脑回路中的性能。 包括树鼩、雪貂和NHP在内的生物。这一提议的结果将是发展， 验证和实施一种新的技术，用于跨突触顺行递送生物制剂， 多个大脑区域和多个物种的代理人。因此，如果成功，我们的项目将最终帮助 开发靶向细胞类型和回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.