Pharmacologic Intervention of PXE Phenotypes

描述（由申请人提供）：弹性假性黄瘤（PXE）是一种遗传性疾病，临床表现为皮肤、眼睛和动脉血管，发病率高，偶有死亡率。PXE是由ABCC6基因突变引起的，大约三分之一的突变是过早终止密码子突变，导致合成截断和无功能蛋白，与无义介导的mRNA衰变（NMD）相关。在这个应用中，我们建议测试已经开发的新的药理学药物，以读取过早终止密码子，从而恢复功能性全长蛋白的合成。这种化合物的原型是PTC124，它已经在其他遗传性疾病的早期临床试验中进行了测试，包括囊性纤维化和肌肉萎缩症，它的药代动力学和毒理学特征已经建立。穿透分子将与NMD拮抗剂结合。本提案将在项目的第一年集中测试在体外细胞培养系统中读透0 - ABCC6无义突变的效率，特别是占所有ABCC6突变约30%的p.R1141X。我们的初步研究已经表明，PTC124诱导p.R1141X终止密码子的读取，导致全长ABCC6蛋白的合成。由于停止密码子已被氨基酸取代，我们将在一种新的斑马鱼mRNA拯救实验中测试全长读通蛋白的功能。在体外研究的同时，我们将继续开发两种新型小鼠模型；(a)突变构建体在肝脏中的瞬时表达，以及(b) ABCC6 -/-背景上p.R1141X突变的人类ABCC6 cDNA敲入小鼠。在项目的第二年，这些模型将在体内测试PTC124的功效。这些小鼠将被喂食含有PTC124的饮食，并结合NMD拮抗剂，如NMDI-1。ABCC6读透的有效性将通过免疫荧光和肝脏特异性抗体的Western分析来检测。PTC124诱导的全长ABCC6合成对ABCC6 -/-小鼠PXE表型的影响将通过外周结缔组织矿化测定、组织病理切片的计算机形态计量学分析和钙磷直接测定来检验。这些研究有望提供PTC124和NMDI-1在对抗PXE矿化表型方面的功效信息。考虑到PXE的患病率约为1:5万，世界上有多达15万人受到影响，其中大多数人有丧失视力和失明的风险。由于大约三分之一的PXE携带无义突变，特别是p.R1141X，在ABCC6基因中，成功解读这些突变将为成千上万的PXE患者提供治疗。

DESCRIPTION (provided by applicant): Pseudoxanthoma elasticum (PXE) is a heritable disease with clinical manifestations in the skin, the eyes, and the arterial blood vessels, with considerable morbidity and occasional mortality. PXE is caused by mutations in the ABCC6 gene, and about a third of all mutations are premature termination codon mutations which result in synthesis of truncated and nonfunctional protein, associated with nonsense-mediated mRNA decay (NMD). In this application, we propose to test novel pharmacological agents that have been developed to read through premature termination codons, thus restoring the synthesis of functional full-length protein. The prototype of such compounds is PTC124 which is already being tested in early clinical trials for other heritable diseases, including cystic fibrosis and muscular dystrophy, and its pharmacokinetics and toxicology profiles have already been established. The read-through molecules will be combined with NMD antagonists. This proposal will concentrate in the first year of the project to test the efficiency of read-through o ABCC6 nonsense mutations, particularly p.R1141X which accounts for ~30% of all ABCC6 mutations, in a cell culture system in vitro. Our preliminary studies have already shown that PTC124 induces read-through of p.R1141X stop codon resulting in synthesis of full-length ABCC6 protein. Since the stop codon has been replaced by an amino acid, we will test the functionality of the full-length read-through protein in a novel zebrafish mRNA rescue assay. In parallel to the in vitro studies, we will proceed with developing two novel mouse models; (a) for transient expression of the mutant construct in the liver, and (b) a knock-in mouse harboring human ABCC6 cDNA with the p.R1141X mutation on the Abcc6-/- background. These models will be tested for the efficacy of PTC124 in vivo during the second year of the project. These mice will be fed with PTC124 containing diet, combined with NMD antagonists, such as NMDI-1. The efficacy of the ABCC6 read-through will be assayed by immunofluorescence and Western analysis of the liver with specific antibodies. The consequences of the synthesis of full-length ABCC6 elicited by PTC124 on the PXE phenotype in Abcc6-/- mice will be examined by assay of mineralization of the peripheral connective tissues by computerized morphometric analysis of histopathologic sections and by direct assay of calcium and phosphorus. These studies are expected to provide information on the efficacy of PTC124 and NMDI-1 in counteracting the mineralization phenotype in PXE. Considering the prevalence of PXE, ~1:50,000, there are as many as 150,000 affected individuals in the world, most of them at risk for loss of vision and for blindness. Since approximately one-third of them harbor nonsense mutations, particularly p.R1141X, in the ABCC6 gene, successful read-through of these mutations would provide treatment for thousands of individuals with PXE.