Cell type specific AAVs to study reward and cognition

腺相关病毒（Adeno-Associated Virus，AAVs）是神经科学研究和基因治疗的有效载体 应用.然而，天然存在的AAV不是细胞类型特异性的：它们必须与其他细胞组合。 技术，如转基因动物，以实现细胞类型特异性基因表达。这一要求限制了 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-并阻碍细胞类型特异性的发展 实现直接临床获益的靶向策略。为了扩大NHP中的细胞类型特异性接入， 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， 特定的AAV。首先，我们将定义细胞类型特异性增强子-远端调控元件， 显示出作为细胞类型特异性AAV驱动程序的相当大的前景。在初步数据中，我们收集了 转录组和染色质可及性（即“多组学”）来自纹状体、背外侧 2只恒河猴的前额叶皮层（dlPFC）、初级运动皮层（M1）、中脑腹侧和中脑。 我们将恒河猴数据集与现有的人类和小鼠数据相结合， 利用神经网络（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其进行排名。 我们将AAV与最佳候选增强子包装在一起，将它们注射到NHP纹状体中，我们观察到， 细胞类型特异性，增强子驱动的纹状体表达-细胞类型特异性纹状体区室相关 奖励处理为了广泛推进这一议程，并开发驱动强大的细胞类型特异性的AAV， 表达，我们建议扩大我们的多组学单细胞数据库的额外数据，从猕猴和 绒猴我们将利用这个更新的、性别平衡的数据库，其中将包括来自8个NHP的数据，以确定 细胞类型特异性增强子，其可能驱动灵长类动物中的稳健表达。与此同时，我们将利用我们的 经验证的scAAVengr管道，用于筛选NHP中细胞类型偏倚感染模式的AAV衣壳突变体 认知和奖励系统。我们将联合收割机将最佳细胞类型特异性增强子与最偏向的AAV 衣壳以产生新的细胞类型特异性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，以创建恒河猴脑图谱，并将验证的载体存储 并由匹兹堡大学生物锻造计划分发。NHP对于研究人类认知至关重要 和疾病，因此迫切需要确定NHP细胞类型的分子特性， 他们的行为功能。该提案将产生一个独特的NHP多组学单细胞数据库，提供细胞 类型特异性AAV的神经元类型的认知和奖励系统，并建立一个新的多模式恒河猴 脑图谱这些贡献将大大提高灵长类动物大脑中的回路操纵能力 促进基础和基础前科学的基础研究。

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.