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

在这里，我们将识别非人类灵长类（NHP）神经元类型，并建立一个广泛的工具箱向量电路- 基于神经科学研究。NHP与NHP在神经解剖学、遗传学和行为学上具有实质性的同源性。 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NHP很重要，但我们缺乏工具， 分析和操纵灵长类动物大脑中的复杂回路。这一缺陷严重限制了基因的使用- 编码的神经科学工具来检查电路的特定功能，并阻碍靶基因的发展 治疗学目前用于在小模式物种中实现转基因的方法，例如创建 基因修饰的菌株在NHP中是极其昂贵的，并且不适用于人类疾病。AAVs 这是生殖系修饰和选择性育种的主要替代方案。AAV感染成年神经元，赋予稳定的 转基因表达，并已在基因治疗临床试验中证明安全。AAV没有天然的细胞类型 特定性质，但当改变或与细胞类型特异性调控序列组合时， （增强子/启动子），它们已经能够实现细胞类型特异性转基因。这使得有可能， 例如，我们之前对中脑多巴胺神经元的学习和决策的光遗传学研究 制作。然而，在AAV介导的基因递送可以推广到整个大脑的回路之前， 多种行为功能，我们必须创造目前缺乏的载体和促进剂，允许有效的， 向所有需要的细胞类型递送特异性基因。在这里，我们将联合收割机单细胞RNA-Seq（scRNA-Seq）与 高通量筛选工程腺相关病毒（AAV），以创建完整的病毒工具箱 载体和启动子，使得能够最小侵入性地监测和操纵NHP脑中的神经元。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 分析并同时筛选腺相关病毒（AAV）载体（衣壳和调控序列） 能够特异性和有效地将转基因递送至分类的神经元。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，其中每个变体与独特的DNA配对， 条形码。然后，我们将scRNA-Seq捕获每个细胞的转录组，并定量AAV和启动子。 每个细胞的表达谱中都有特定的条形码。在恒河猴身上进行的初步实验 验证并展示了这种创新方法的前景。我们的具体目标的结果将 包括（1）视网膜、前额叶皮层、初级运动皮层和纹状体中的细胞类型的清单，（2）细胞 靶向所有定义的细胞类型的类型特异性AAV和启动子，（3）具有广泛向性的AAV，（4）公共靶向的AAV。 数百万NHP脑细胞转录谱的可用数据集，（5）更新和全面的 恒河猴参考基因组，和（6）细胞类型的解剖学、生理学和功能验证- 特定的电路工具及其在NHP大脑中的功能。

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.