Microglia-Integrated Brain Organoids for Gaucher Disease Modeling and Therapeutic Discovery - Summary Neuronopathic Gaucher disease (nGD) is a severe pediatric genetic disorder caused by mutations in GBA1, the gene that codes for lysosomal acid β-glucosidase (GCase). Defective GCase impairs the degradation of glucosylceramide (GluCer) and glucosylsphingosine (GluSph) in the central nervous system (CNS), activating glial cells and contributing to neuroinflammation. The prevalence of GD is 1 in 500 among Ashkenazi Jews and 1 in 60,000 in the general population. Approximately 10% of GD patients in the US and Europe, and around 75% in Asian countries, are diagnosed with nGD. Patients with nGD often manifest early in life with high mortality. Emerging evidence implicates microglial dysfunction in the pathogenesis of various neurodegenerative disorders, including nGD, but the mechanisms by which GCase deficiency leads to microglial activation and neurodegeneration in nGD remain largely unknown. Current enzyme replacement therapy (ERT) and substrate reduction therapy (SRT) are ineffective for CNS involvement, and their safety for newborns is not well understood. GD is a hereditary lysosomal storage disorder that is included in newborn screening programs, allowing for early detection and timely intervention. Traditional models, including 2D cultures fail to replicate the complexity of human brain tissues. Utilizing induced pluripotent stem cells (iPSCs) derived from nGD patients, we have successfully developed nGD midbrain-like organoids (MLOs) model. These nGD organoid models exhibit midbrain characteristics and nGD-specific phenotypes, such as GCase deficiency and GluSph accumulation, providing a model system for studying nGD and a platform for drug evaluation. Furthermore, we have advanced MLOs by incorporating microglia to create microglia-containing MLOs (mcMLOs). Microglial differentiation was achieved by inducing the homeostatic transcription factor PU.1 in re-engineered iPSCs within a neural differentiation environment, leading to the formation of mcMLOs. In GD, GluCer and GluSph accumulation activates microglia, increasing cytokine release and inflammation via the C5a/C5aR1 pathway. We hypothesize that microglia within mcMLOs profoundly influence nGD-relevant phenotypes, including microglial activation, and that the complement C5a/C5a receptor system plays a role in exacerbating neuroinflammation. Specifically blocking C5aR1 signaling in GCase-deficient microglia may represent a therapeutic target to prevent microglia- mediated inflammation and neurodegeneration in nGD. To test this hypothesis, in Aim 1, we will generate nGD mcMLOs to investigate the impact of nGD microglia in MLO growth and disease phenotypes, and to evaluate therapeutic effects on this nGD mcMLO model. In Aim 2, we will determine the role of the microglial C5a/C5aR1 pathway in nGD and assess C5aR1 inhibition as a therapeutic approach. This advanced nGD mcMLO model offers a unique, physiologically relevant platform using patient iPSC-derived organoids, enabling mechanistic studies and the development of novel therapies for nGD.
