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成了人类大脑的功能回路。世纪以来，神经科学家 脑细胞的分类是根据它们的特征，包括形状，位置，生理学，分子， 功能单细胞转录组学研究现在以前所未有的分辨率定义分子细胞类型 这是可能的，揭示了数百至数千种脑细胞类型的分类。这些研究还揭示了 物种间同源细胞类型的分子特征的巨大差异，决定性地表明， 老鼠和人类大脑的区别不仅仅是神经元的总数。然而，函数 大脑回路中每种细胞类别或类型的功能，以及疾病中的功能障碍，才刚刚开始被评估。到 为了描述人脑细胞类别在正常功能和疾病中的作用， 被开发来允许基因进入体内的细胞类别。这些工具将使精确的治疗基因 递送到脑细胞类别，允许针对类别特异性病因（如一些癫痫）的靶向治疗。 很少有遗传工具可用于标记和操纵非遗传易处理的细胞类别和类型。 人类和非人类灵长类动物（NHP）。病毒包括腺相关病毒（AAV）， 含有细胞类别和类型选择性增强子的细胞可以被利用来获得对基因的遗传接近，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BRAIN Initiative发起了一个项目， 产生和验证报告AAV以在体内和人类中标记小鼠皮层中的特定细胞类别 离体新皮层组织。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中的许多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展，但一个障碍是， 在灵长类动物中验证新载体的过程既昂贵又耗时。 我们提出了三个目标，将这些有前途的新AAV载体转化为一组高价值的灵长类动物优化的载体。 这些工具最终可以用于人类的基因治疗。首先，我们将开发一个筛选平台 NHP离体脑切片中的AAV载体，随后在体内NHP中对有希望的载体进行单独验证 和人离体脑切片培养物。其次，我们将鉴定最佳的AAV衣壳以： 当静脉内或脑脊液（CSF）应用时，体内NHP神经元转导，两种优选的 用于人CNS基因治疗的递送途径，和B）支持人原代脑组织的AAV转导 离体。第三，我们将使用细胞类别选择性载体进行概念验证实验，以表达 治疗性转基因在定义的类别，以治疗一种严重的和难治性的形式，儿童癫痫， Dravet综合征（DS）。这些实验代表了将细胞类别选择性 将AAV转化为一流的病毒工具，优化用于体内NHP脑研究和人类基因治疗应用。

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.