Unlocking microglia targeting for neurotherapeutics

项目摘要/摘要 小胶质细胞是大脑和脊髓中长期驻留的巨噬细胞，它不断地 调查微环境，以便对伤害和感染做出快速反应。不像神经元或其他 胶质细胞和小胶质细胞是可移植的，这使它们成为靶向治疗的理想对象。 神经系统疾病。创造和改进这种治疗方法的一个主要障碍是无法 用慢病毒对小胶质细胞进行基因工程。这项提案旨在识别和克服 小胶质细胞限制因子，以实现慢病毒的基因编辑并深入了解 小胶质细胞介导的抗病毒免疫。目标1将检验假设cGAS/STING，a 已知的抗病毒途径，是使用抑制剂和转基因技术限制小胶质细胞病毒的基础 小鼠小胶质细胞的体外实验，用一种新的小胶质细胞特异的Cre小鼠进行体内验证。在AIM 2，我将使用新创建的无偏小胶质细胞聚焦CRISPR基因敲除屏幕 具有小胶质细胞嗜性的AAVs，以发现防止慢病毒感染的新的抗病毒途径。 目的2展示了在原代小胶质细胞中的第一个CRISPR屏幕。这种早期的独立 应用程序为我的独立研究项目提供了一个愿景。它基于一个平台 我在过去十年中创建了基于小胶质细胞的发现工具，并在我作为 儿科神经科医生，照顾患有神经免疫疾病的儿童，包括 脑白质营养不良和干扰素病。这将为基因靶向研究提供新的途径。 小胶质细胞用于神经治疗，小胶质细胞抗病毒免疫的直接测试，并将影响 未来的研究方向与HIV潜伏期的神经后遗症和 神经免疫性疾病。

PROJECT SUMMARY/ABSTRACT Microglia are the long-lived resident macrophages of the brain and spinal cord, which constantly survey the microenvironment to respond rapidly to injury and infection. Unlike neurons or other glia, microglia are transplantable, making them ideal candidates for targeted therapies to treat neurological diseases. A major barrier to creating and refining such treatments is an inability to genetically engineer microglia with lentiviruses. This proposal aims to identify and overcome microglia restriction factors to enable gene editing with lentiviruses and gain insights into microglia-mediated antiviral immunity. Aim 1 will test the hypothesis that cGAS/STING, a known anti-viral pathway, underlies microglial viral restriction using inhibitors and transgenic mouse microglia in vitro, with in vivo validation using a new microglia-specific Cre mouse. In Aim 2, I will perform an unbiased microglia-focused CRISPR knockout screen using newly created AAVs with microglia tropism to discover new anti-viral pathways that prevent lentiviral infection. Aim 2 demonstrates the first CRISPR screen in primary microglia. This Early Independence application offers a vision for my independent research program. It is based on a platform of microglia-based discovery tools I created over the last decade, and driven by my work as a pediatric neurologist who cares for children with neuroimmunological conditions including leukodystrophies and interferonopathies. It will provide new approaches to genetic targeting of microglia for neurotherapeutics, direct testing of microglia antiviral immunity, and will influence future directions related to the neurological sequelae of HIV latency and the pathogenesis of neuroimmunological disease.