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

摘要 该提案的总体目标是表征酸性鞘磷脂酶（aSMase）在鞘脂中的功能 为了研究体内代谢和病理生物学，并开发更精确的酶替代疗法（ERT）， 尼曼-匹克病（NPD）。ASMase催化鞘磷脂（SM）水解为神经酰胺， 磷酸胆碱aSM酶功能障碍导致NPD A型和B型，一种溶酶体储存疾病 其特征在于鞘磷脂在内溶酶体隔室中积聚（1）。NPD患者- A发展为严重的神经和内脏病理学，很少能活超过3岁（2）， NPD-B通常活到青春期/成年早期，没有神经系统体征或症状的表现（3）。 最近对使用aSMase蛋白或质粒用于重组蛋白或DNA治疗的努力的兴趣已经增加。 与非人类灵长类动物的炎症增加有关（4）。这是因为SMPD 1基因 其编码aSM酶，产生两种不同的酶-溶酶体鞘磷脂酶（L-SM酶）和 分泌型鞘磷脂酶（S-SMase），通过一个共同的蛋白质前体的差异运输。我们 合作者先前在细胞中证明，aSMase中的Ser 508 Ala（S508 A）突变 （aSMaseS 508 A）保留L-SMase活性，但S-SMase有缺陷（5）。此外，我们已经证明， 表达aSMaseS 508 A突变体的细胞中S-SMase活性的丧失阻止了趋化因子的扩增， 炎性细胞因子（6）。先前的工作已经证明，表达aSMase融合蛋白的小鼠 保留L-SMase活性的细胞表现出对小脑浦肯野细胞层的保护作用， aSMase缺陷小鼠观察到严重的神经系统疾病（7）。因此，仔细确定在 S508 A突变体的体内功能可能允许其发展为缺乏 炎症效应。 基于这些数据，我们的实验室已经产生了一种新的转基因小鼠模型（GEMM）， SMPD 1中的S508 A点突变。该GEMM aSMaseS 508 A是与杰克逊合作生成的 使用CRISPR-Cas9技术的实验室。我们在这些小鼠中的初步数据表明， 血清中S-SMase活性。 因此，这项建议的目标是创新和重要的，因为这将是第一项研究， 定义这种SMPD 1变体在体内的作用，定义这种突变对鞘脂的影响， NPD的代谢、病理和症状。为此，我们提出以下具体目标： 具体目标1。确定aSMaseS 508 A突变对体内鞘脂代谢的影响。 具体目标2。定义aSMaseS 508 A对体内NPD病理生物学的影响。

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.