An enhancer-AAV toolbox to target and manipulate distinct interneuron subtypes [P28]

Establishing tools that can target and manipulate specific neuronal populations is critical to understanding neural circuits and developing targeted therapies. Recombinant adeno-associated virus (rAAV) is a popular tool for gene delivery and is widely used in basic research as well as gene therapy. Significant effort has been made to develop rAAV tools that can drive cell-type or tissue specific transgene expression. One of the strategies to develop such tools is to incorporate cell-type specific enhancer elements into the rAAV genome. In recent years, we and others have identified a number of enhancers that, when incorporated into rAAV vectors, can restrict the transgene expression to particular neuronal populations. Yet, viral tools to access and manipulate fine neuronal subtypes are still limited. Here, we performed systematic analysis of single cell ATAC-seq and RNA-seq data to identify enhancer candidates for each of the cortical interneuron subtypes.We established a set of enhancer-AAV tools that are highly specific for distinct cortical interneuron populations and striatal cholinergic neurons. These enhancers, when used in the context of different effectors, can target (fluorescent proteins), observe activity (gCaMP) and manipulate (opto- or chemo-genetics) specific neuronal subtypes. We also validated our enhancer-AAV tools across species. Thus, we provide the field with a powerful set of tools to study neural circuits and functions and to develop precise and targeted therapy. To identify potential cell type specific enhancers, we took advantage of the single-cell assay for transposase accessible chromatin using sequencing (scATAC-seq) data we have previously generated in the lab. To enrich for GABAergic cells, Dlx6a-Cre::INTACT mice were used. GFP+ nuclei from cortex (anterior lateral motor cortex, ALM) and striatum were isolated by FACS and scATAC-seq data were collected

研发能够靶向并操纵特定神经元群体的工具，对于解析神经环路以及开发靶向治疗手段至关重要。重组腺相关病毒（Recombinant adeno-associated virus, rAAV）是一类主流的基因递送工具，被广泛应用于基础研究与基因治疗领域。学界已投入大量精力研发可驱动细胞类型或组织特异性转基因表达的rAAV工具。开发此类工具的策略之一，是将细胞类型特异性增强子元件整合至rAAV基因组中。近年来，本团队与其他研究团队均已筛选出多类增强子，将其整合至rAAV载体后，可将转基因表达限定于特定神经元群体中。然而，可精准靶向并操纵精细神经元亚型的病毒工具仍较为匮乏。本研究对单细胞ATAC测序（single-cell Assay for Transposase-Accessible Chromatin using sequencing, scATAC-seq）与RNA测序数据展开系统性分析，旨在筛选针对各皮层中间神经元亚型的候选增强子。我们成功构建了一套增强子-AAV工具，可对不同皮层中间神经元群体以及纹状体胆碱能神经元实现高特异性靶向。将这些增强子与不同效应元件配合使用时，可实现对特定神经元亚型的标记（荧光蛋白）、活性观测（gCaMP）以及操纵（光遗传学或化学遗传学）。我们还跨物种验证了这套增强子-AAV工具的有效性。综上，本研究为领域内提供了一套强大的工具集，可用于神经环路与功能研究，以及开发精准靶向的治疗方案。为筛选潜在的细胞类型特异性增强子，我们利用了本实验室此前生成的单细胞转座酶可及性染色质测序（single-cell assay for transposase-accessible chromatin using sequencing, scATAC-seq）数据。为富集γ-氨基丁酸能（GABAergic）神经元，本研究使用了Dlx6a-Cre::INTACT小鼠模型。通过荧光激活细胞分选（Fluorescence Activated Cell Sorting, FACS）分离获得皮层（前外侧运动皮层，anterior lateral motor cortex, ALM）与纹状体中的GFP阳性细胞核，并完成scATAC-seq数据采集。