Study On The PTC124Treatment For Pseudoxanthoma Elasticum

Research Background and ObjectivesPseudoxanthoma Elasticum (PXE) is an autosomal recessive metabolic disorderwith estimated prevalence of1:50,000, but with no current effective treatment. PXE iscaused by mutations in the ABCC6gene, and more than300different mutations areknown to cause PXE, more than35%of these being nonsense mutations.Methods1. Study on HEK293cell in vitroWe investigated the therapeutic potential of the premature termination codon (PTC)readthrough-inducing drug PTC124in treating PXE in an in vitro system. The ability ofdrug to readthrough PTC was examined in HEK293cells transfected with sevendifferent ABCC6nonsense DNA-constructs linked to a DDK peptide tag at the3’ endassociated with PXE. The efficacy of the read-through was evaluated byimmunofluorescence and In-Cell ELISA at~72hours after treatment with the drug..2. Study on zebrafish model in vivoConsidering the redundancy of the genetic code, it was postulated that in case ofp.R1141X, the read-through may result in substitution of the arginine1141by glycine,tryptophan or cysteine. The potential pathogenicity of these three missense substitutionswas tested in a novel zebrafish mRNA rescue assay.ResultsPTC124did not exhibit toxicity at concentrations up to40μg/ml and the highestreadthrough efficiency was observed at dosage of5μg/ml when a series of doses weretested within a range from0to40μg/ml. In addition, PTC124at5μg/ml was moreefficient on R1164X and R1275X than on other mutants tested, including R1141X,indicating that induced readthrough is not only a dose-dependent but also a context-dependent process. The zebrafish mRNA rescue study suggested that all threemRNA transcripts were able to rescue Abcc6morpholino-induced mortality ofdeveloping zebrafish embryo, similar to wild-type mRNA, indicating that they are notpathogenic.Conclusions1. PTC124enhances the read-through efficiency of premature termination codonmutation of ABCC6gene. Higher efficiency was observed with5-10ug/ml, a dosewithout cell toxicity.2. Readthrough is not only a dose-dependent but also a context-dependent process3. These3subsitution of mRNA transcripts were able to rescue Abcc6morpholino-induced mortality of developing zebrafish embryo, indicating that they arenot pathogenic.4. We set up an evaluation system working well not only for the PTC124andaminoglycoside catalog, but also for the more and more novel drugs in development totreat the nossense mutation in PXE and other genetic dermatologic disease.This studyprovides valuable information concerning the potential of pharmacological treatment forPXE.