Pharmacologic Intervention of PXE Phenotypes

描述（由申请人提供）：弹性假黄瘤（PXE）是一种遗传性疾病，临床表现为皮肤、眼睛和动脉血管，发病率相当高，偶尔死亡。PXE是由ABCC 6基因突变引起的，约三分之一的突变是提前终止密码子突变，导致合成截短和无功能的蛋白质，与无义介导的mRNA衰变（NMD）相关。 在本申请中，我们建议测试已开发的用于读取提前终止密码子的新型药理学试剂，从而恢复功能性全长蛋白质的合成。这种化合物的原型是PTC 124，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营养不良症，其药代动力学和毒理学特征已经建立。通读分子将与NMD拮抗剂组合。 该提案将集中在项目的第一年，以测试ABCC 6无义突变的通读效率，特别是在体外细胞培养系统中占所有ABCC 6突变的约30%的p.R1141X。我们的初步研究已经表明，PTC 124诱导p.R1141X终止密码子的通读，导致全长ABCC 6蛋白的合成。由于终止密码子已被氨基酸取代，我们将在一种新的斑马鱼mRNA拯救试验中测试全长通读蛋白的功能。 在体外研究的同时，我们将继续开发两种新的小鼠模型;（a）用于突变体构建体在肝脏中的瞬时表达，以及（B）在Abcc 6-/-背景上携带p.R1141 X突变的人ABCC 6 cDNA的敲入小鼠。这些模型将在项目的第二年进行PTC 124体内疗效测试。这些小鼠将用含有PTC 124的饮食与NMD拮抗剂如NMDI-1组合喂养。ABCC 6通读的功效将通过免疫荧光和具有特异性抗体的肝脏的Western分析来测定。由PTC 124引发的全长ABCC 6的合成对Abcc 6-/-小鼠中的PXE表型的影响将通过测定外周结缔组织的矿化来检查，所述测定通过组织病理学切片的计算机化形态测定分析和通过钙和磷的直接测定来进行。这些研究有望提供关于PTC 124和NMDI-1在抵消PXE中矿化表型的功效的信息。 考虑到PXE的患病率约为1：50，000，世界上有多达150，000人受影响，其中大多数人面临视力丧失和失明的风险。由于大约三分之一的人在ABCC 6基因中含有无义突变，特别是p.R1141X，这些突变的成功通读将为数千名患有PXE的个体提供治疗。

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 Since the stop codon has been replaced by an amino acid, we will test the functionality of the full-length read-through protein in a novel zebrafish mRNA rescue assay.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.