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

开发动物模型以将溶酶体与酸性鞘磷脂酶的炎症功能分离

• 批准号: 10133427
• 负责人: Ashley J. Snider
• 金额: $ 7.54万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10133427
• 关键词: Developing; animal; models; dissociate; lysosomal

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.

抽象的 该提案的总体目标是表征鞘脂中酸鞘磷脂酶（ASMase）的功能 代谢和病理学在体内，并开发更精确的酶替代疗法（ERT） Niemann-Pick病（NPD）。 ASMASE将鞘磷脂（SM）的水解催化至神经酰胺和 磷胆碱。 ASMase的功能障碍导致NPD型A和B，溶酶体存储障碍 其特征是在内溶液室内积聚鞘磷脂（1）。 NPD-患者 发育严重的神经和内脏病理学，很少居住在3岁以上（2），而患者 NPD-B通常生活到青少年/成年早期，没有神经系统征兆或症状的表现（3）。 最近对使用ASMase蛋白或质粒进行重组蛋白或DNA疗法的努力的兴趣已 与非人类隐私的感染增加有关（4）。这是因为SMPD1基因 编码ASMase，产生两种不同的酶 - 溶酶体鞘磷脂酶（L -Smase）和 秘书长鞘磷脂酶（S-Smase），通过对共同蛋白质前体的差异运输。我们的 合作者以前在细胞中证明了ASMase中的Ser508Ala（S508A）突变 （ASMASES508A）保留L-SMase活性，但在S-Smase中有缺陷（5）。此外，我们已经证明了 表达ASMASES508A突变体的细胞中S-SMase活性的丧失可防止趋化因子扩增 炎性细胞因子（6）。先前的工作表明，表达ASMase融合蛋白的小鼠 保留L-Smase活性暴露了小脑Purkinje细胞层的保护，并受到保护 严重的神经系统疾病观察到ASMase缺乏小鼠（7）。因此，仔细确定 S508A突变体的体内功能可以使其发展为有效的ERT（或基因置换） 炎症作用。 在这些数据的基础上，我们的实验室生成了一种新颖的经过修改的鼠标模型（GEMM），其中包含 SMPD1中的S508A点突变。这个Gemm Asmases508a是与杰克逊合作生成的 实验室使用CRISPR – CAS9技术。这些小鼠中我们的初步数据显示 血清中的S-Smase活性。 因此，该提案的目标具有创新性和意义，因为这将是第一个直接的研究 定义该SMPD1变体在体内的作用，从而定义了该突变对鞘脂的影响 NPD的代谢，病理和症状。为此，我们提出以下具体目的： 具体目标1。建立Asmases508a突变对体内鞘脂代谢的影响。 具体目标2。定义Asmases508a对体内NPD病理生物学的影响。