Engineered viral tropism for cell-type specific manipulation of neuronal circuits

 描述（由申请人提供）：这是神经科学的一个长期目标，揭示特定的细胞类型如何有助于不同的神经回路，这些神经回路是认知，行为和疾病病理的基础。虽然细胞类型可以分为描述性类别（兴奋性、抑制性、肽能等），我们知道，在离子通道和受体表达水平上不同并在神经回路中发挥离散作用的神经元有很大的组合多样性。因此，为了提高神经回路图的分辨率，为了理解大脑如何在机械水平上工作，以及为了更好地理解疾病病理，非常需要操纵神经回路中更特定的细胞集。当将转基因传递到许多神经元时，遗传靶向这些不同的子集是困难的（具有潜在的不利影响）和依赖于细胞类型特异性启动子进行选择性表达-当前的技术状态。我们的议程是从根本上改变如何实现细胞类型特异性遗传操作：由于神经元的功能定义--它对刺激的电生理反应--本质上是一个蛋白质组学问题，我们提出了一种新的病毒递送方法，该方法能够选择性地将转基因递送到在细胞表面上表达一组靶向离子通道和受体的神经元。当使用这种新方法时，转基因表达可以由通用和可靠的启动子或其他工程化启动子系统（例如对光或药物敏感）驱动。为了实现这一变革性目标，即为体内病毒基因递送提供一种广泛有用的工具，我们利用施密特博士在使用遗传编码肽毒素的蛋白质工程方面的专业知识，以及托马斯博士在成瘾性疾病体内模型方面的专业知识。在本申请中，我们描述了一种可推广的方法的发展，用于创建具有用户可选择的向性的工程病毒，其可以靶向神经元细胞类型的特定子集。我们还提出在完整的脑组织中证明这些工程病毒的实用性，包括光遗传学靶向-不依赖于转基因动物或特异性启动子-参与奖励相关突触可塑性的两组神经元。这项工作的成果将是一种广泛有用的一流病毒递送技术，该技术能够根据它们表达的表面受体对大脑中定义的神经元类型进行遗传操作。这种方法将使探索神经活动的分子和细胞机制的全新方法成为可能。

 DESCRIPTION (provided by applicant): It is a longstanding goal in neuroscience to reveal how specific cell types contribute to different neural circuits that underlie cognition, behavior, and disease pathology. Although cell types can be grouped into descriptive categories (excitatory, inhibitory, peptidergic etc.), we know there is a great combinatorial diversity of cels that differ in ion channel and receptor expression levels and fulfill discrete roles within neural circuits. Thus, to improve the resolution of neural circuit maps, to understand how the brain works on a mechanistic level, and to better understand disease pathologies there is a great need for manipulating ever more specific sets of cell in neural circuits. Genetically targeting these different subsets is difficult when delivering transgenes to many neurons (with potentially adverse effects) and relying on cell-type specific promoters for selective expression - the current state of the art. Our agenda is to fundamentally change how cell type specific genetic manipulation is achieved: Since the functional definition of a neuron - its electrophysiological response to a stimulus - is intrinsically a proteomic problem, we propose a novel viral delivery method able to deliver transgenes selectively to neurons that express, on the cell surface, a targeted set of ion channels and receptors. When using this novel method transgene expression can be driven from generic and reliable promoters or other engineered promoter systems (e.g. sensitive to light or drugs). To achieve this transformative goal of a broadly useful tool for in vvo viral gene delivery, we build on Dr. Schmidt's expertise in protein engineering using genetically encoded peptide toxins, and Dr. Thomas' expertise with in vivo models of addiction disorders. In this application we describe the development of a generalizable method for creating engineered viruses with user-selectable tropism that can target specific subsets of neuronal cell types. We furthermore propose to demonstrate utility of these engineered viruses in intact brain tissue, including optogenetically targeting - without relying on transgenic animals or specific promoters - two sets of neurons involved in reward-related synaptic plasticity. The outcome of this work will be a broadly useful and first-in-class viral delivery technology that enables the genetic manipulation of defined sets neuron types in the brain based on what surface receptors they express. This method will enable completely new ways of exploring molecular and cellular mechanism of neural activity.