Developing animal models to dissociate lysosomal from inflammatory functions of acid sphingomyelinase

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Abstract The overall goal of this proposal is to characterize the function of acid sphingomyelinase (aSMase) in sphingolipid metabolism and pathobiology in vivo and to develop more precise enzyme replacement therapy (ERT) for Niemann-Pick disease (NPD). ASMase catalyzes the hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine. Dysfunction of aSMase results in NPD types A and B, a lysosomal storage disorder characterized 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 with NPD-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 have been associated with increased inflammation in non-human primates (4). This is because the SMPD1 gene which encodes aSMase, gives rise to two distinct enzymes - lysosomal sphingomyelinase (L-SMase) and secretory sphingomyelinase (S-SMase), via differential trafficking of a common protein precursor. Our collaborators 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 that loss 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 protein that retained L-SMase activity exhibited protection of the cerebellar Purkinje cell layer and were protected from the severe neurologic disease observed aSMase deficient mice (7). Therefore, careful determination of the in vivo function of the S508A mutant may allow its development as effective ERT (or gene replacement) devoid of inflammatory effects. Building on these data, our lab has generated a novel genetically modified mouse model (GEMM) containing the S508A point-mutation in SMPD1. This GEMM, aSMaseS508A, was generated in collaboration with Jackson Laboratories using CRISPR–Cas9 technology. Our preliminary data in these mice demonstrate complete loss of S-SMase activity in serum. Therefore, the goals of this proposal are innovative and significant as this will be the first study to directly define the role of this SMPD1 variant in vivo, defining the effects of this mutation on sphingolipid metabolism, 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.