Cell type selective viral tools to interrogate and correct non-human primate and human brain circuitry

摘要： 许多类型的细胞共同组装了人脑的功能回路。一个多世纪以来，神经学家 根据特征对脑细胞类型进行分类，包括形状、位置、生理、分子和 功能。单细胞转录学研究现在以一种以前不同的分辨率来定义分子细胞类型 有可能，揭示出成百上千种脑细胞类型的分类。这些研究还揭示了 不同物种同源细胞类型的分子签名的显著差异，明确表明 老鼠和人脑的区别不仅仅是神经元的总数。但是，该函数 大脑回路中每种细胞类别或类型，以及疾病中的功能障碍，才刚刚开始评估。至 为了描述人类脑细胞类别在正常功能和疾病中的作用，至关重要的是工具 开发的目的是允许基因进入体内的细胞类别。这样的工具将使精确的治疗基因 输送到脑细胞类别，允许针对特定类别的病因进行有针对性的治疗，如一些癫痫。 很少有遗传工具可以用来标记和操作非遗传易处理的细胞类别和类型 像人类和非人灵长类(NHP)这样的物种。病毒包括腺相关病毒(AAV)， 含有细胞类和类型选择性增强子可以被用来获得遗传途径，并驱动基因 在这些物种的特定脑细胞类别中表达。我们通过大脑倡议发起了一个项目 制备和验证报告AAVs以标记活体和人类小鼠皮质中的特定细胞类别 体外培养的新皮质组织。我们的团队已经设计了AAV载体并优化了衣壳以访问神经元 并在小鼠和灵长类动物的多种离散细胞类别和类型中表达转基因。新的和改进的AAV 工具有望推动人类大脑的科学发现和临床进步，但其中一个障碍是 在灵长类动物中验证新媒介的过程既昂贵又耗时。 我们提出了三个目标来将这些有前景的新AAV载体转化为灵长类优化的高价值集合 这些工具最终可能被用于人类的基因治疗。首先，我们将开发一个筛选平台 NHP体外脑片中的AAV载体，随后在NHP体内对有前景的载体进行个体验证 和人类体外脑片培养。第二，我们将确定最佳的AAV衣壳以：a)支持广泛的 体内NHP神经元转导在静脉或脑脊液中应用时，首选两种 人类中枢神经系统基因治疗的递送途径，以及b)支持人原代脑组织的AAV转导 体外实验。第三，我们将使用细胞类选择向量来执行概念验证实验，以表达 特定类别的治疗性转基因治疗一种严重的、难治性的儿童癫痫，称为 Dravet综合征(DS)。这些实验代表着朝着转化细胞类别选择性迈出的重要一步 AAVS成为一流的病毒工具，针对体内NHP脑研究和人类基因治疗应用进行了优化。

Abstract: Many cell types together assemble the functional circuitry of the human brain. For over a century, neuroscientists have categorized brain cell types by their features, including shape, position, physiology, molecules, and function. Single cell transcriptomics studies are now defining molecular cell types at a resolution not previously possible, uncovering a taxonomy of hundreds to thousands of brain cell types. These studies have also revealed dramatic differences in molecular signatures of homologous cell types across species, showing decisively that the difference between mouse and human brain is not simply the total number of neurons. However, the function of each cell class or type in brain circuitry, and dysfunction in disease, is only beginning to be evaluated. To characterize the roles of human brain cell classes in normal function and disease, it is critical that tools be developed to allow genetic access to cell classes in vivo. Such tools would enable precise therapeutic gene delivery to brain cell classes, permitting targeted treatment for class-specific etiologies like some epilepsies. Few genetic tools are available to mark and manipulate cell classes and types in non-genetically tractable species like human and non-human primate (NHP). Viruses including adeno-associated viruses (AAVs), containing cell class and type selective enhancers can be leveraged to gain genetic access to, and drive gene expression in specific brain cell classes in these species. We have initiated a project through the BRAIN Initiative to generate and validate reporter AAVs to mark specific cell classes in the mouse cortex in vivo and in human neocortical tissue ex vivo. Our groups have engineered AAV vectors and optimized capsids to access neurons and express transgenes in many discrete cell classes and types in mouse and primate. New and improved AAV tools promise to fuel human brain scientific discovery and clinical progress, but one impediment has been the costly and time-consuming process of validating new vectors in primates. We present three Aims to translate these promising new AAV vectors into a high-value set of primate-optimized tools that could eventually be used for gene therapies in humans. First, we will develop a platform for screening AAV vectors in NHP ex vivo brain slices, followed by individual validation of promising vectors in NHP in vivo and human ex vivo brain slice cultures. Second, we will identify optimal AAV capsids to: a) support widespread NHP neuronal transduction in vivo when applied intravenously or to cerebrospinal fluid (CSF), two preferred routes of delivery for human CNS gene therapy, and b) support AAV transduction of human primary brain tissue ex vivo. Third, we will perform proof-of-concept experiments using cell class-selective vectors to express a therapeutic transgene in defined classes to treat a severe and intractable form of childhood epilepsy called Dravet syndrome (DS). These experiments represent a significant step towards converting cell class-selective AAVs into first-in-class viral tools optimized for in vivo NHP brain studies and human gene therapy applications.