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

针对转录定义的灵长类细胞类型的庞大 AAV 库

基本信息

批准号：
9804256
负责人：
William Richard Stauffer
金额：
$ 228.7万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-09-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9804256
关键词：
Adult Anatomy Behavior Behavioral Brain Brain region Breeding Capsid Cells Clinical Code Cognition Cognitive Complex Consensus Corpus striatum structure DNA Data Set Databases Decision Making Dependovirus Development Dimensions Disease Dopamine Receptor Engineering Enhancers Equipment and supply inventories Evolution Expression Profiling Foundations Gene Delivery Gene Expression Gene Expression Profile Gene Transfer Techniques Genes Genetic Genetic Transcription Genomics Goals Human Individual Investigation Investigational Therapies Learning Libraries Macaca Macaca mulatta Maps Mediating Mental disorders Methods Midbrain structure Modeling Modification Monitor Monkeys Moods Motor Cortex Mus Mutate Neurologic Neurons Neurosciences Opsin Outcome Photophobia Photosensitivity Physiological Prefrontal Cortex Primates Property Regulatory Element Retina Retinal Degeneration Retinal Ganglion Cells Retinal gene therapy Rewards Role Specificity Structure System Thalamic structure Transgenes Tropism Tyrosine 3-Monooxygenase Update Validation Variant Viral Vector Vision adeno-associated viral vector base brain cell cell type cost dopaminergic neuron experimental study functional restoration gene therapy clinical trial high throughput screening human disease innovation minimally invasive multidisciplinary nervous system disorder neural circuit nonhuman primate optogenetics promoter reference genome relating to nervous system single-cell RNA sequencing synthetic biology therapeutic gene therapeutic transgene tool transcriptome transgene expression validation studies vector visual processing

项目摘要

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.

在这里，我们将识别非人类灵长类动物 (NHP) 神经元类型，并为电路构建一个广泛的向量工具箱- 基于神经科学研究。 NHP 与 NHP 具有大量的神经解剖学、遗传和行为同源性人类，因此他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电路。这种缺乏严重限制了基因技术的使用编码神经科学工具来检查电路特定功能并阻碍目标基因的发育疗法。当前在小型模型物种中实现转基因的方法，例如创建转基因菌株在 NHP 中价格昂贵且不适用于人类疾病。 AAV 是种系修饰和选择性育种的领先替代方案。 AAV 感染成年神经元，赋予稳定的转基因表达，并在基因治疗临床试验中证明是安全的。 AAV 没有天然细胞类型特定的特性，但当改变或与细胞类型特定的调控序列组合时（增强子/启动子）他们已经能够实现细胞类型特异性转基因。这使得，例如，我们之前对中脑多巴胺神经元进行学习和决策的光遗传学研究制作。然而，在 AAV 介导的基因传递能够推广到大脑回路并用于治疗之前，多种行为功能，我们必须创造目前缺乏的载体和启动子，以允许有效和将特定基因递送至所有所需的细胞类型。在这里，我们将单细胞 RNA-Seq (scRNA-Seq) 与高通量筛选工程腺相关病毒 (AAV)，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方法根据单个神经元的基因表达对其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）能够将转基因特异性且有效地递送至分类的神经元。首先我们要综合大量的突变 AAV 载体和合成启动子文库，其中每个变体都与独特的 DNA 配对条形码。然后我们将通过 scRNA-Seq 捕获每个细胞的转录组并量化 AAV 和启动子 - 每个细胞表达谱中都有特定的条形码。恒河猴的初步实验已完全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清单，(2) 细胞针对所有定义的细胞类型的类型特异性 AAV 和启动子，(3) 具有广泛趋向性的 AAV，(4) 公开的数百万 NHP 脑细胞的可用转录谱数据集，(5) 更新且全面的恒河猴参考基因组，以及（6）细胞类型的解剖学、生理学和功能验证- NHP 大脑中的特定电路工具及其功能。