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（AAVS）是神经科学研究和基因疗法的潜在基因递送向量申请。但是，自然发生的AAV并非特定于细胞类型：它们必须与其他技术，例如转基因动物，以实现细胞类型特异性基因表达。此要求限制使用通常编码的“断路”工具来研究非人类素数（NHP）的行为 - 与人类相似最大的实验动物模型，并阻碍细胞类型的发展针对达到直接临床益处的策略。在NHP中扩展特定于单元格的特定访问，然后放置电路特定基因治疗的基础，我们建议创建，测试和验证下一代，细胞类型特定的AAV。首先，我们将定义特定细胞类型的增强子 - 具有的远端调节元素作为特定于细胞类型的AAV驱动程序，证明了相当大的希望。在初步数据中，我们收集了转录组和染色质可及性（即“多摩变”）单细胞数据来自纹状体，背外侧前额叶皮层（DLPFC），原发性运动皮层（M1），岛岛和腹中脑的2个恒河猕猴。我们将恒河猴数据集与现有的人类和鼠标数据相结合，并使用了卷积神经网络（CNN）根据其作为细胞类型特异性增强剂的潜力来对开放染色质序列进行排名。我们将AAV与顶级候选增强剂打包，并将其注入NHP纹状体，我们观察到细胞类型的特异性，增强子驱动的链球菌 - 一种细胞类型特异性纹状体室与奖励处理。为了广泛推进这一Agernda并开发出驱动鲁棒，特定于细胞类型的AAVS 表达式，我们建议通过Macaque和果果。我们将利用此更新的性能均衡数据库，其中包含来自8个NHP的数据以识别细胞类型特定的增强剂可能会驱动私人表达强劲的表达。同时，我们将使用我们的经过验证的Scaavengr管道筛选AAV CAPSID突变体，用于NHP中的细胞类型偏置感染模式认知和奖励系统。我们将将特异性特异性增强剂与最有偏见的AAV结合在一起 Capsids生成新的细胞类型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。这些数据将与超高分辨率MRI扫描以创建恒河猕猴的脑图，并将存储经过验证的向量并由匹兹堡Bioforge大学倡议分发。 NHP对于研究人类认知至关重要和疾病，因此需要定义NHP细胞类型的分子特性并进行研究他们的行为功能。该建议将生成独特的NHP多摩尼克单细胞数据库，提供单元格在认知和奖励系统中为神经元类型键入特定的AAV，并建立新的多模式恒河脑图集。这些贡献将显着提高初级大脑的电路操纵能力并促进基础和临床前科学领域的基本研究。