Pharmacologic Intervention of PXE Phenotypes

描述(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，偶尔会死亡。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，导致截短和无功能蛋白的合成，与无义介导的mRNA衰退(NMD)相关。在这个应用中，我们建议测试新的药理药物，这些药物已经被开发来阅读提前终止密码子，从而恢复功能全长蛋白质的合成。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测试，包括囊性纤维化和肌营养不良症，它的药代动力学和毒理学特征已经建立。通读的分子将与NMD拮抗剂结合。这项建议将集中在项目的第一年，以测试在体外细胞培养系统中ABCC6无义突变的通读效率，特别是占所有ABCC6突变的30%的p.R1141X。我们的初步研究已经表明，PTC124可以诱导P.R1141X终止密码子的通读，从而合成全长的ABCC6蛋白。由于终止密码子已经被氨基酸取代，我们将在一种新的斑马鱼mRNA救援试验中测试全长通读蛋白的功能。在体外研究的同时，我们将继续开发两个新的小鼠模型：(A)突变结构在肝脏中的瞬时表达，(B)在Abcc6-/-背景上带有p.R1141X突变的人ABCC6基因的敲入小鼠。在项目的第二年，这些模型将在体内测试PTC124的有效性。这些小鼠将被喂以含有PTC124的食物，并结合NMD拮抗剂，如NMDI-1。ABCC6通读的有效性将通过免疫荧光和带有特定抗体的肝脏的Western分析来检测。PTC124诱导的全长ABCC6的合成对Abcc6-/-小鼠PXE表型的影响将通过组织病理切片的计算机形态计量分析和直接测定钙和磷的外周结缔组织矿化分析来检验。这些研究有望提供有关PTC124和NMDI-1在抑制PXE矿化表型方面的有效性的信息。考虑到PXE的流行率，约1：50,000，世界上有多达150,000人受到影响，其中大多数人面临失明和失明的风险。由于其中约三分之一存在无义突变，特别是ABCC6基因中的p.R1141X，这些突变的成功通读将为数千名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 Since the stop codon has been replaced by an amino acid, we will test the functionality of the full-length read-through protein in a novel zebrafish mRNA rescue assay.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.