Cell type specific AAVs to study reward and cognition

腺相关病毒(AAV)是神经科学研究和基因治疗的有效基因载体 申请。然而，自然产生的AAVs不是特定于细胞类型的：它们必须与其他 转基因动物等技术，以实现细胞类型特定基因的表达。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的行为-- 与人类最相似的实验动物模型-并阻碍特定细胞类型的发展 有针对性的策略，以实现直接的临床益处。在NHP中扩展特定于细胞类型的访问，并为 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 特定的AAV。首先，我们将定义细胞类型特定的增强子-具有 展示了相当大的前景作为细胞类型特定的AAV驱动器。在初步数据中，我们收集了 纹状体背外侧的转录和染色质可及性(即“多组”)单细胞数据 2只恒河猴的前额叶皮质(DlPFC)、初级运动皮质(M1)、脑岛和腹侧中脑。 我们将恒河猴的数据集与现有的人类和小鼠数据结合起来，并使用卷积神经 网络(CNN)根据其作为细胞类型特异性增强子的潜力对开放的染色质序列进行排序。 我们用最好的候选增强剂包装动静脉动静脉，将它们注射到NHP纹状体，我们观察到 细胞类型特异的、增强子驱动的纹状体表达--一种细胞类型特异的纹状体间隔与 奖励处理。要广泛推进这一议程并开发出强大的、特定于细胞类型的AAVs 表达，我们建议扩展我们的多染色体单细胞数据库，使用来自猕猴和 一只绒猴。我们将利用这个更新的性别平衡数据库，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，可能在灵长类动物中驱动强劲的表达。同时，我们将使用我们的 验证scAAVengr管道在NHP中筛选AAV衣壳突变体的细胞类型偏向感染模式 认知系统和奖励系统。我们将结合顶级细胞类型特定增强子和最偏向的AAV 衣壳生成新的细胞类型特异性AAVs，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)来验证AAV的特异性。该数据将与 超高分辨率核磁共振扫描创建恒河猴脑图谱，验证后的载体将被存储 并由匹兹堡大学生物锻造计划分发。NHP是研究人类认知的关键 和疾病，因此迫切需要确定NHP细胞类型的分子特性并研究 它们的行为功能。该方案将生成一个独特的NHP多组单细胞数据库，提供细胞 认知和奖赏系统中神经元类型的特定AAVs，并建立新的多通道恒河猴 脑图集。这些贡献将显著提高灵长类大脑的电路操作能力。 推进基础科学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.