Abstract Body: Lysosomal storage disorders (LSDs) are genetic metabolic diseases with lysosomal enzyme deficiencies. Deficiency of or defective acid sphingomyelinase (ASM) causes Niemann-Pick disease types A and B (NPDA and NPDB, respectively), neurological disorders characterized by accumulation of sphingomyelin. We developed a nanodisc (ND)-based platform wherein recombinant ASM has been embedded in a bilayer of phospholipids circumscribed by apolipoprotein E3 (apoE3) for targeted delivery to the lysosomes. The rationale is that apoE3 would serve as a high-affinity ligand via its N-terminal (NT) domain for the low-density lipoprotein receptors (LDLr) facilitating receptor-mediated endocytosis of the nanodisc; the nanodisc and its components would eventually disintegrate in the low-pH environment of the lysosomes thereby releasing the embedded ASM. We designed a fusion protein comprising the essential catalytic domain of ASM flanked by the transmembrane domain (TMD) of a pulmonary surfactant protein at the N-terminal end and a His-tag at the C-terminal end to facilitate purification. The fusion construct termed TMD-ASM was expressed and purified from a bacterial expression system, and the identity confirmed by Western blot using anti-ASM and anti-His tag antibodies. TMD-ASM was reconstituted with phospholipids and apoE3 NT to promote formation of lipoprotein complexes, followed by density gradient ultracentrifugation to isolate nanodiscs with ASM (TMD-ASM-ND). Empty ND without the embedded ASM were generated in parallel. Physicochemical characterization using SDS-PAGE shows the presence of both ASM (35kDa) and apoE3NT (24 kDa) in the nanodisc preparations, while native PAGE revealed the formation of large complexes (~600 kDa) of ~ 15 nm diameter. Electron microscopy imaging of TMD-ASM-ND revealed nanodisc formation, while atomic force microscopy showed nanodisc ~15 nm high. Co-immunoprecipitation confirmed the presence of ASM in nanodiscs, while sphingomyelinase activity assays revealed robust ASM activity of TMD-ASM-ND. Lastly, glioblastoma cells, which are known to overexpress LDLr were treated with TMD-ASM-ND and the cellular uptake followed by immunofluorescence. Fluorescence microscopy revealed punctate perinuclear vesicles with co-localization of ASM with apoE3 and lysosomal marker, indicative of successful targeting of ASM to the lysosomes. These findings show potential for targeted delivery of ASM using nanodiscs to treat NPD by enzyme replacement therapy.