AbstractThe overall goal of this proposal is to characterize the function of acid sphingomyelinase (aSMase) in sphingolipidmetabolism and pathobiology in vivo and to develop more precise enzyme replacement therapy (ERT) forNiemann-Pick disease (NPD). ASMase catalyzes the hydrolysis of sphingomyelin (SM) to ceramide andphosphocholine. Dysfunction of aSMase results in NPD types A and B a lysosomal storage disordercharacterized by accumulation of sphingomyelin within the endolysosomal compartment (1). Patients with NPD-A develop severe neurologic and visceral pathology and rarely live beyond 3 years of age (2) while patients withNPD-B typically live to adolescence/early adulthood with no manifestation of neurological signs or symptoms (3).Recent interest in the efforts to use aSMase proteins or plasmids for recombinant protein or DNA therapy havebeen associated with increased inflammation in non-human primates (4). This is because the SMPD1 genewhich encodes aSMase gives rise to two distinct enzymes - lysosomal sphingomyelinase (L-SMase) andsecretory sphingomyelinase (S-SMase) via differential trafficking of a common protein precursor. Ourcollaborators have previously demonstrated in cells that the Ser508Ala (S508A) mutation in aSMase(aSMaseS508A) retains L-SMase activity but is defective in S-SMase (5). Furthermore we have demonstrated thatloss of S-SMase activity in cells expressing the aSMaseS508A mutant prevents chemokine amplification by pro-inflammatory cytokines (6). Previous work has demonstrated that mice expressing an aSMase fusion proteinthat retained L-SMase activity exhibited protection of the cerebellar Purkinje cell layer and were protected fromthe severe neurologic disease observed aSMase deficient mice (7). Therefore careful determination of the invivo function of the S508A mutant may allow its development as effective ERT (or gene replacement) devoid ofinflammatory effects. Building on these data our lab has generated a novel genetically modified mouse model (GEMM) containingthe S508A point-mutation in SMPD1. This GEMM aSMaseS508A was generated in collaboration with JacksonLaboratories using CRISPRCas9 technology. Our preliminary data in these mice demonstrate complete loss ofS-SMase activity in serum.Therefore the goals of this proposal are innovative and significant as this will be the first study to directlydefine the role of this SMPD1 variant in vivo defining the effects of this mutation on sphingolipidmetabolism pathology and symptoms of NPD. To this end we propose the following specific aims:Specific Aim 1. Establish the effects of the aSMaseS508A mutations on sphingolipid metabolism in vivo.Specific Aim 2. Define the effects of aSMaseS508A on NPD pathobiology in vivo.