Cell type specific AAVs to study reward and cognition

用于研究奖励和认知的细胞类型特异性 AAV

基本信息

批准号：
10517904
负责人：
Leah Byrne
金额：
$ 685.02万
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：
2022
资助国家：
美国
起止时间：
2022-09-01 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10517904
关键词：
Anatomy Animal Model Atlases Bar Codes Behavioral Biological Assay Brain Callithrix Capsid Cell Nucleus Cells Chromatin Clinical Clinical Trials Code Cognition Cognitive Collaborations Corpus striatum structure DNA Data Data Set Databases Dependovirus Development Disease Distal Engineering Enhancers Experimental Animal Model Fluorescent in Situ Hybridization Foundations Gene Delivery Gene Expression Gene Transfer Techniques Human Image Infection Injections Insula of Reil Knowledge Laboratory Animals MRI Scans Macaca Macaca mulatta Magnetic Resonance Imaging Midbrain structure Molecular Molecular Analysis Monkeys Motor Cortex Multiomic Data Mus Neurons Neurosciences Nootropic Agents Outcome Pattern Prefrontal Cortex Primates Production Property Regulatory Element Reporter Research Personnel Resolution Rest Rewards Rhesus Science Specificity Structure System Techniques Technology Testing Time Transgenic Animals United States National Institutes of Health Universities Update Vocabulary Work adeno-associated viral vector base behavioral study cell type comparative genomics computerized tools convolutional neural network delivery vehicle fundamental research gene therapy genomic tools molecular subtypes multimodality multiple omics mutant neurotropic next generation nonhuman primate pre-clinical prevent reward processing sex striosome success targeted treatment tool transcriptomics translational applications ultra high resolution vector web based interface

项目摘要

Adeno-Associated Viruses (AAVs) are potent gene delivery vectors for neuroscience studies and gene therapy applications. However, naturally occurring AAVs are not cell type specific: they must be combined with other technologies, such as transgenic animals, to achieve cell type specific gene expression. This requirement limits the use genetically coded ‘circuit-breaking’ tools to study behavior in nonhuman primates (NHPs) – the experimental animal model with the greatest similarity to humans – and hinders development of cell type specific targeting strategies for achieving direct clinical benefits. To expand cell type specific access in NHPs and lay the foundation for circuit specific gene therapy, we propose to create, test, and validate next generation, cell type specific AAVs. First, we will define cell type specific enhancers – distal regulatory elements that have demonstrated considerable promise as cell type specific AAV drivers. In preliminary data, we collected transcriptomic and chromatin accessibility (i.e. “multi-omic”) single cell data from the striatum, dorsolateral prefrontal cortex (dlPFC), primary motor cortex (M1), insula, and ventral midbrain of 2 rhesus macaque monkeys. We combined the rhesus monkey data set with existing human and mouse data and used convolutional neural networks (CNNs) to rank open chromatin sequences according to their potential as cell type specific enhancers. We packaged AAVs with the top candidate enhancers, injected them into NHP striatum, and we have observed cell type specific, enhancer driven expression in striosomes – a cell type specific striatal compartment related to reward processing. To broadly advance this agenda and develop AAVs that drive robust, cell type-specific expression, we propose to expand our multi-omic single cell database with additional data from macaque and marmoset. We will leverage this updated, sex-balanced database, with will include data from 8 NHPs to identify cell type specific enhancers that are likely to drive robust expression in primates. In parallel, we will use our validated scAAVengr pipeline to screen AAV capsid mutants for cell-type biased infection patterns in the NHP cognitive and reward systems. We will combine the top cell type specific enhancers with the most biased AAV capsids to generate new, cell type specific AAVs for targeting neurons in the NHP cognitive and reward systems. We will validate AAV specificity using Fluorescent in situ hybridization (FISH). This data will be combined with ultra-high resolution MRI scans to create a rhesus macaque brain atlas, and the validated vectors will be stored and distributed by The University of Pittsburgh BioForge Initiative. NHPs are critical for studying human cognition and disease, and thus there is a pressing need to define the molecular properties of NHP cell types and study their behavioral functions. This proposal will generate a unique NHP multi-omic single cell database, provide cell type specific AAVs for neuron types in cognitive and reward systems, and establish a new multimodal rhesus brain atlas. These contributions will significantly advance circuit manipulation capabilities in the primate brain and promote fundamental research in basic and preclinical science.

腺相关病毒 (AAV) 是用于神经科学研究和基因治疗的有效基因传递载体然而，天然存在的 AAV 不具有细胞类型特异性：它们必须与其他细胞结合。技术，例如转基因动物，以实现细胞类型特异性基因表达。使用基因编码的“断路”工具来研究非人类灵长类动物（NHP）的行为与人类最相似的实验动物模型 - 并且阻碍特定细胞类型的发育扩大 NHP 中特定细胞类型的使用范围并奠定基础。作为电路特异性基因治疗的基础，我们建议创建、测试和验证下一代细胞类型首先，我们将定义细胞类型特异性增强子——具有的远端调节元件。在初步数据中，我们收集了作为细胞类型特异性 AAV 驱动程序的巨大前景。来自纹状体、背外侧的转录组和染色质可及性（即“多组学”）单细胞数据两只恒河猴的前额皮质 (dlPFC)、初级运动皮质 (M1)、岛叶和腹侧中脑。我们将恒河猴数据集与现有的人类和小鼠数据相结合，并使用卷积神经网络网络（CNN）根据开放染色质序列作为细胞类型特异性增强子的潜力对它们进行排名。我们将 AAV 与顶级候选增强子一起包装，将它们注射到 NHP 纹状体中，我们观察到纹状体中细胞类型特异性、增强子驱动的表达——与以下相关的细胞类型特异性纹状体区室广泛推进这一议程并开发驱动强大的、特定于细胞类型的 AAV。表达式，我们建议使用来自猕猴和的额外数据来扩展我们的多组学单细胞数据库我们将利用这个更新的、性别平衡的数据库，其中包括来自 8 个 NHP 的数据来识别。同时，我们将使用我们的细胞类型特异性增强子，这些增强子可能会在灵长类动物中驱动强大的表达。经验证的 scAAVengr 管道可筛选 AAV 衣壳突变体，以了解 NHP 中细胞类型偏向的感染模式我们将把顶级细胞类型特异性增强剂与最有偏见的 AAV 结合起来。衣壳产生新的、细胞类型特异性的 AAV，用于靶向 NHP 认知和奖励系统中的神经元。我们将使用荧光原位杂交 (FISH) 验证 AAV 特异性。该数据将与该数据相结合。超高分辨率 MRI 扫描创建恒河猴大脑图谱，并将存储经过验证的矢量由匹兹堡大学 BioForge Initiative 分发的 NHP 对于研究人类认知至关重要。和疾病，因此迫切需要定义 NHP 细胞类型的分子特性并进行研究该提案将生成一个独特的NHP多组学单细胞数据库，提供细胞。针对认知和奖励系统中神经类型的特定 AAV，并建立新的多模式恒河猴这些贡献将显着提高灵长类动物大脑的电路操纵能力。促进基础和临床前科学的基础研究。