Acid Sphingomyelinase and Niemann-Pick Disease

描述（由申请人提供）： 这项研究的总体目标是利用我们独特的 A 型和 B 型尼曼-匹克病 (NPD) 小鼠模型来开发针对这种疾病的新疗法并揭示新的致病机制。提出了两个目标。目标 1：两种新疗法的临床前评估。在第一组研究中，我们将评估一种使用热休克蛋白 Hsp70 的新型酶增强方法。这些实验基于最近发表的一篇文章，该文章显示 Hsp70 增强了 A 型和 B 型 NPD 患者细胞中残留的 ASM 活性并减少了溶酶体病理学，这一发现得到了自上次提交以来获得的新体内数据的支持。第二组研究将继续我们的工作，旨在使用 ICAM-1 改善 ASM 向肺部的输送。将进行“原理验证”基因转移实验，使用 AAV8 载体在 NPD 小鼠的肝脏中表达 ASM/ICAM-1 融合蛋白。将研究融合酶在肺和其他临床重要器官中的吸收和功效。新的体内数据也证明了这种方法的可行性。目标 2：研究导致 ERT 相关肝脏毒性的新致病机制。 ASM 敲除 (ASMKO) 小鼠的临床前 ERT 研究表明，以高于 5 mg/Kg 的剂量施用重组 ASM 会导致肝出血、肝酶释放和死亡。我们最近发现，这些小鼠肝脏中主要积聚的脂质是鞘氨醇，而不是鞘磷脂，并推测鞘氨醇的储存至少部分是造成 ERT 相关毒性的原因。我们将通过评估 ASMKO 小鼠中神经酰胺酶、鞘氨醇激酶和 1-磷酸鞘氨醇裂解酶的表达来确定累积鞘氨醇的来源，并将这些动物与缺乏鞘氨醇激酶 1 的小鼠交配，以确定对 ERT 相关肝脏毒性的影响。这些结果应该为小鼠的临床观察提供机制基础，并且还可能导致克服这种毒性的新策略。

DESCRIPTION (provided by applicant): The overall goal of this research is to use our unique mouse models of Types A & B Niemann-Pick disease (NPD) to develop new therapies for this disorder and to uncover novel pathogenic mechanisms. Two aims are proposed. Aim 1: Preclinical Evaluation of Two New Therapies. In the first set of studies we will evaluate a novel enzyme enhancement approach using the heat shock protein, Hsp70. These experiments are based on a recent publication showing that Hsp70 enhanced residual ASM activity and reduced lysosomal pathology in cells from Type A and B NPD patients, a finding that has been supported by new in vivo data obtained since the previous submission. The second set of studies will continue our work aimed at improved delivery of ASM to the lung using ICAM-1. A "proof-of-principle" gene transfer experiment will be undertaken that will use AAV8 vectors to express ASM/ICAM-1 fusion proteins in the livers of NPD mice. Uptake and efficacy of the fusion enzymes in the lung and other clinically important organs will be studied. New in vivo data also has been obtained to demonstrate the feasibility of this approach. Aim 2: Investigation of Novel Pathogenic Mechanisms Leading to ERT-Related Liver Toxicity. Preclinical ERT studies in ASM knockout (ASMKO) mice have shown that administration of recombinant ASM at doses above 5 mg/Kg results in liver bleeding, the release of liver enzymes, and death. We have recently found that sphingosine, not sphingomyelin, is the major accumulating lipid in the livers of these mice, and hypothesize that sphingosine storage is responsible, at least in part, for the ERT-associated toxicity. We will determine the source of accumulating sphingosine by evaluating the expression of ceramidases, sphingosine kinases and sphingosine-1-phosphate lyase in the ASMKO mice, and breed these animals to mice lacking sphingosine kinase-1 to determine the effects on ERT- associated liver toxicity. These results should provide a mechanistic basis for the clinical observations in the mice, and also may lead to new strategies to overcome this toxicity.