SapC-based Brain-targeting Delivery of Long-acting Enzymes for Neuronopathic Gaucher Disease Therapy - Summary Gaucher disease (GD) is caused by mutations in the GBA1 gene encoding a lysosomal enzyme, acid β- glucosidase (GCase). GD is classified as visceral (Type 1) or neuronopathic (Types 2 and 3, nGD) diseases. Defective GCase function results in the gradual buildup of its substrates glucosylceramide (GluCer) and glucosylsphingosine (GluSph), leading to conditions such as hepatosplenomegaly, chronic anemia, thrombocytopenia, and osteopenia in Type 1, or the severe neuronopathic forms (Types 2 and 3, nGD) which often manifest early in life with high mortality. Prevalence of GD in Ashkenazi Jews is 1/500 and, in the general population, 1/60,000. Approximately 10% of GD patients in the US and Europe, and around 75% in Asian countries, are diagnosed with nGD. The outcomes of visceral manifestation in GD patients are improved by Enzyme Replacement Therapy (ERT), but ongoing limitations include treatment delivery regimen challenges and high costs in part due biological instability of GCase requiring high doses to maintain a therapeutic effect. Moreover, and pertinent to this proposal, ERT is ineffective for nGD due to the blood-brain barrier (BBB) blocking central nervous system (CNS) access. Consequently, the CNS disease in nGD patients is currently untreatable. To enable GCase delivery of a more biologically stable form through the BBB into the CNS, we are developing an innovative approach that 1) utilizes a distinctive and safe nanocarrier for GCase consisting of Saposin C (SapC) and dioleoylphosphatidylserine (DOPS) that has the capability to cross the BBB and 2) uses a novel GCase (named fGCase) with markedly improved stability. Our preliminary data have shown the following features supporting the therapeutic promise of SapC-DOPS-fGCase nanodrug: 1) increased plasma half-life activity, 2) enhanced stability in both cells and the brain after crossing the BBB, 3) generalized entry of fGCase throughout the brain, 4) sustained catalytical activity in the brain as evidenced by a ~29% reduction in GluCer and a ~44% reduction in GluSph levels after a single intravenous dose over five days. The preliminary results strongly indicate that SapC-DOPS-fGCase, exhibiting remarkable stability in the brain and extended catalytic activity, has the potential to provide sustained and functional enzymatic action that could effectively prevent phenotypic deficits in nGD patients who currently lack viable treatment options. To demonstrate in vivo efficacy of SapC-DOPS-fGCase for further development to treat human nGD in the R61 phase, in Aim 1, we will optimize the formulation of SapC-DOPS with fGCase, validate its stability, and, in Aim 2, characterize pharmacokinetic properties to establish a dosing regimen. Upon demonstration of sustainable stability and activity of SapC-DOPS- fGCase and establishing therapeutic dosing regimen, in the R33 phase, Aim 3, we will evaluate in vivo efficacy of SapC-DOPS-fGCase in a nGD mouse model for treating nGD. Development of the novel SapC-DOPS-fGCase nanodrug will radically improve the delivery of more stable longer-acting enzymes to the brain, providing a promising CNS-ERT for patients with nGD.
