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 Since the stop codon has been replaced by an amino acid, we will test the functionality of the full-length read-through protein in a novel zebrafish mRNA rescue assay.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.
Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, manifests with cutaneous, ocular and vascular findings, with considerable morbidity and mortality. There is no specific or effective treatment for the systemic manifestations of this disease. This project focuses on pre-clinical development of pharmacological approaches to counteract PXE in a subset of patients with premature termination codon mutations in the ABCC6 gene.
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