Directed Evolution of Novel AAVs and Regulatory Elements for Selective Microglial Gene Expression

项目摘要 小胶质细胞炎症与许多神经系统疾病的病理学有关，包括 神经发育障碍，如自闭症和唐氏综合征;神经生成障碍，如 阿尔茨海默病（AD）、帕金森病、肌萎缩侧索硬化（ALS）和亨廷顿病; 和神经性疼痛。利用腺相关病毒（AAV）载体的基因治疗已经成为一种高度有效的方法。 治疗中枢神经系统（CNS）疾病的有前途的策略，以及免疫抑制基因 因此，抑制小胶质细胞中的免疫信号传导途径的疗法将非常有希望用于治疗这种广泛的 一系列慢性疾病。然而，这种信号通路在其他CNS细胞中起保护作用，包括 因此，治疗传递不仅需要是有效的，而且需要靶向小胶质细胞。 通过利用我们在病毒工程、单细胞分析、机器学习以及人类和 非人类灵长类动物模型，我们建议开发一个技术平台， 在成年灵长类动物的大脑中，特别是小胶质细胞。我们将把AAV的定向进化与分子生物学结合起来， 条形码、单细胞下一代测序（NGS）、机器学习以及人类组织和非人类组织 灵长类动物（NHP）脑模型以开发用于选择性递送至灵长类动物小胶质细胞的AAV。此外，为了进一步 为了增强这些技术的特异性，我们将应用类似的文库选择，NGS和机器 学习方法来设计短的合成启动子，并确定选择性增强子的内源性增强子。 小胶质细胞基因表达。最后，这些能力将被应用于提供潜在的治疗基因货物 to microglia小胶质细胞in vitro体外and in vivo体内. 总之，我们提出了一个高风险，创新的研究计划，如果成功，将提高我们的能力 选择性地调节小胶质细胞中的免疫信号，如果成功的话， 广泛的神经系统疾病此外，这项工作将建立一种广泛影响的技术， 一个集成了矢量工程、下一代测序和机器学习的平台， 用于细胞特异性遗传操作，其原则上可以应用于细胞中的任何细胞或组织。 中枢神经系统或身体。因此，我们期望我们经验丰富的多学科团队能够提供强大的 对技术发展、神经科学以及其他领域的基础和转化生物学的贡献 系统.

Project Summary Microglial inflammation has been implicated the pathology of a host of neurological conditions, including neurodevelopmental disorders such as autism and Down Syndrome; neurogenerative disorders such as Alzheimer's disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and Huntington’s disease; and neuropathic pain. Gene therapy utilizing adeno-associated viral (AAV) vectors has emerged as a highly promising strategy for treating central nervous system (CNS) disorders, and an immunosuppressive gene therapy to inhibit immune signaling pathways in microglia would thus be highly promising for treating this broad range of chronic conditions. However, this signaling pathway serves protective roles in other CNS cells including neurons, such that therapeutic delivery would need to be not only efficient but targeted to microglia. By leveraging our expertise in viral engineering, single cell analysis, machine learning, and human and non-human primate models, we propose to develop a technology platform for genetically accessing specific cell types in the adult primate brain, in particular microglia. We will integrate directed evolution of AAV with molecular barcoding, single cell next generation sequencing (NGS), machine learning, and human tissue and non-human primate (NHP) brain models to develop AAVs for selective delivery to primate microglia. Additionally, to further enhance the specificity of these technologies, we will apply analogous library selection, NGS, and machine learning approaches to engineer short, synthetic promoters and to identify endogenous enhancers for selective microglial gene expression. Finally, these capabilities will be applied to deliver potential therapeutic gene cargoes to microglia in vitro and in vivo. In sum, we propose a high-risk, innovative research program that will, if successful, advance our capacity to selectively modulate immune signaling in microglia, work that if successful will have implications for treating a broad range of neurological conditions. Furthermore, this work will establish a broadly impactful technology platform that integrates vector engineering, next generation sequencing, and machine learning to engineer tools for cell specific genetic manipulation, which can in principle be applied to in principle any cell or tissue in the central nervous system or body. We thus anticipate that our experienced, multidisciplinary team can offer strong contributions to technology development, neuroscience, and fundamental and translational biology in other systems.