Summary Gaucher disease (GD) is a common lysosomal storage disease, occurring at a rate of 1 in 500 in Ashkenazi Jews and 1/60,000 in the general population. Defective acid β-glucosidase (GCase) in GD results in progressive accumulation of glycolipid substrates in tissues leading to hepatosplenomegaly, weakened bone, impaired blood clotting and neurological impairments. Despite many advances in GD therapy, treatments are only partially effective for the more common visceral forms and are completely inadequate for a highly symptomatic and frequently lethal subtype of the disease, neuronopathic GD (nGD), affecting the central nervous system (CNS). Current enzyme replacement treatments (ERT) for nGD are limited by two major challenges: limited penetration of the blood-brain barrier (BBB) and instability of GCase of existing ERT resulting in short half-lives in circulations and organs. We have developed a novel Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanocarrier that can penetrate the BBB. When SapC-DOPS is combined with a novel stable enzyme, named fGCase, allows for efficient transport into the brain with sustained bioactivity. Our intravenously delivered, long-acting SapC-DOPS- fGCase retains kinetic stability in mouse plasma and cells, penetrates through the BBB into the CNS, and displays prolonged activity leading to reduction of brain-accumulated glycolipid substrates. Preliminary results suggest that interplay between SapC, phosphatidyl serine (PS), a receptor for SapC, and the lymphatic system influences SapC-DOPS’s ability to transport the enzyme across the BBB and process in the brain. These preliminary findings strongly suggest that the highly brain-stable SapC-DOPS-fGCase will maintain adequate and sustainable activities to restore GCase function in GD brains. Based on these promising features of SapC- DOPS and fGCase, our overall hypothesis is that SapC-DOPS-fGCase will reestablish GCase function in GD brains and improve brain disease outcomes to advance enzyme treatment for nGD. In Aim 1, we will 1) determine the pharmacokinetics and biodistribution of SapC-DOPS-fGCase, 2) evaluate the therapeutic impact in nGD mouse models and human nGD iPSC-derived midbrain-like organoid model, and 3) investigate the underlying mechanism(s) of this novel CNS-ERT approach in protecting neuronal cell functions. In Aim 2, we will define the pathways involved in the transport of SapC-DOPS-fGCase across the BBB. Our focus will be to investigate the interplay of cell surface PS and SapC-DOPS and the role of CNS-lymphatics for SapC-DOPS-fGCase processing in the brain. Completing this preclinical study will provide proof of concept for SapC-DOPS-fGCase to be a transformative ERT for human nGD. Additionally, we will gain a deeper understanding of PS-dependent, CNS-targeting of SapC-DOPS through the CNS-lymphatic system.