A Massive Library of AAVs to Target Transcriptionally-Defined Primate Cell Types

在这里，我们将识别非人灵长类(NHP)神经元类型并构建一个广泛的向量工具箱，用于电路- 基于神经科学的研究。NHP在神经解剖学、遗传学和行为方面与 人类，因此它们对于研究认知和设计的神经回路基础是不可或缺的 治疗神经和精神疾病的疗法。尽管卫生保健计划很重要，但我们缺乏工具来 分析和操纵灵长类动物大脑中的复杂电路。这种缺乏严重限制了基因- 编码神经科学工具来检查电路特定功能并阻碍靶向基因的发展 治疗学。目前在小型模式物种中实现转基因的方法，例如创造 转基因菌株，在NHP中昂贵得令人望而却步，不适用于人类疾病。AAV是 种系改良和选育的领先替代品。AAVs感染成年神经元，提供稳定的 转基因表达，并已被证明是安全的基因治疗临床试验。动静脉动静脉没有天然的细胞类型 特定的性质，但当改变或与细胞类型特定的调控序列结合时 (增强剂/启动子)他们已经能够实现针对细胞类型的转基因。这使得， 例如，我们之前对学习和决策的中脑多巴胺神经元的光遗传学研究 制作。然而，在AAV介导的基因传递可以推广到整个大脑和大脑的回路之前， 多重行为功能，我们必须创造目前缺乏的载体和启动子，允许高效和 特定的基因传递给所有所需的细胞类型。这里，我们将单细胞RNA-Seq(scRNA-Seq)与 高通量筛选工程化腺相关病毒(AAV)以创建完整的病毒工具箱 载体和启动子能够对NHP大脑中的神经元进行微创监测和操作。我们 设计了一种跨学科的方法，根据基因表达对单个神经元进行分类 同时筛查腺相关病毒载体(衣壳和调控序列) 能够向分类的神经元提供特定和高效的转基因。首先，我们将合成大量的 突变的AAV载体和合成启动子的文库，其中每个变体都与唯一的DNA配对 条形码。然后，我们将使用scRNA-Seq捕获每个细胞的转录组，并对AAV和启动子进行定量- 每个细胞的表达配置文件中的特定条形码。在恒河猴身上的初步实验已经充分 验证并展示了这种创新方法的前景。我们具体目标的结果将是 包括(1)视网膜、前额叶皮质、初级运动皮质和纹状体的细胞类型清单，(2)细胞 针对所有定义的细胞类型的特定类型的AAVs和启动子，(3)具有广泛取向的AAVs，(4)公开的AAVs 可获得数百万NHP脑细胞转录图谱的数据集，(5)更新和全面的 恒河猴参考基因组，以及(6)细胞类型的解剖、生理和功能验证- NHP大脑中特定的电路工具及其功能。

Here we will identify nonhuman primate (NHP) neuron types and build an extensive toolbox of vectors for circuit- based neuroscience studies. NHPs share substantial neuroanatomical, genetic, and behavioral homology with humans, and therefore they are indispensable for investigating the neural circuit basis of cognition and devising therapies to treat neurological and psychiatric disorders. Despite the importance of NHPs, we lack the tools to analyze and manipulate complex circuits in the primate brain. This lack severely limits the use of genetically- coded neuroscience tools to examine circuit specific functions and hinders development of targeted gene therapeutics. Current methods for achieving transgenesis in small model species, such as the creation of genetically modified strains, are prohibitively expensive in NHP and not applicable to human disease. AAVs are the leading alternative to germline modification and selective breeding. AAVs infect adult neurons, confer stable transgene expression, and have proven safe in gene therapy clinical trials. AAVs do not have natural cell-types specific properties, but when altered or combined with cell type specific regulatory sequences (enhancers/promoters) they have been able to achieve cell type-specific transgenesis. This has made possible, for example, our previous optogenetic investigation of midbrain dopamine neurons for learning and decision making. However, before AAV-mediated gene delivery can be generalized to circuits across the brain and for multiple behavioral functions, we must create currently lacking vectors and promoters that permit efficient and specific gene delivery to all required cell types. Here, we will combine single-cell RNA-Seq (scRNA-Seq) with high-throughput screening of engineered adeno-associated viruses (AAVs) to create a complete toolbox of viral vectors and promoters enabling minimally invasive monitoring and manipulation of neurons in NHP brain. We have devised a transdisciplinary approach to classify individual neurons according to their gene expression profile and simultaneously screen for adeno-associated virus (AAV) vectors (capsids and regulatory sequences) capable of specific and efficient transgene delivery to classified neurons. First, we will synthesize massive libraries of mutated AAV vectors and synthetic promoters, in which each variant is paired with a unique DNA barcode. We will then scRNA-Seq to capture the transcriptome for each cell and quantify the AAV and promoter- specific barcodes in every cell’s expression profile. Preliminary experiments in Rhesus monkeys have fully validated and demonstrated the promise of this innovative approach. The outcomes of our Specific Aims will include (1) an inventory of cell types in the retina, prefrontal cortex, primary motor cortex, and striatum, (2) cell type-specific AAVs and promoters targeting all defined cell types, (3) AAVs with broad tropisms, (4) a publicly available dataset of transcription profiles for millions of NHP brain cells, (5) an updated and comprehensive Rhesus macaque reference genome, and (6) anatomical, physiological, and functional validation of cell type- specific circuits tools and their function in the NHP brain.