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.

Project Narrative Microglial inflammation has been implicated in the pathology of numerous neurodegenerative diseases, neurodevelopmental disorders, and other neurological conditions, and suppressing the activation of innate immune signaling pathways in these cells could thus represent a broad therapeutic approach. To enhance the efficacy and safety of such an approach, the intervention should be both long-term and targeted to microglia, such that a gene therapy based on an adeno- associated viral (AAV) vector would be promising. Since natural AAVs exhibit inefficient, non-targeted delivery to human microglia, however, we propose to 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 gene delivery of potential therapeutic cargoes to primate microglia.