6.3.1. PTC-124

About 400 distinct disease-causing variants have so far been reported in the *ABCC6* gene (https://www.ncbi.nlm.nih.gov/clinvar, accessed on 3 February 2021). A little more than 1/3 of all variants are nonsense that most likely result in nonsense-mediated decay or possibly truncated ABCC6 proteins. The most common and recurrent variant (and the first to be identified) is the nonsense p.R1141X, which accounts for ∼30% of all pathogenic alleles in PXE patients of Caucasian descent [169,170].

Correct protein synthesis is at the core of biological processes of all living organisms. Therefore, many compounds with the ability to overcome premature termination codons (PTC) have been studied over the years with the hope of restoring full-length protein synthesis as a therapeutic approach for heritable diseases [171]. Indeed, about 12% of human genetic disorders are caused by nonsense mutations, leading to the generation of PTC. This strategy was tried previously with cystic fibrosis patients. In a phase II clinical trial, these patients were given 1,2,4-oxadiazole (PTC-124), a non-aminoglycoside nonsense mutation suppressor, with some success [172]. However, follow-up phase III trials were relatively unsuccessful [173]. This drug is approved for clinical use by the European Medicines Agency (EMA) to treat Duchenne muscular dystrophy but not by the Federal Drug Administration in the US. It is commercialized under the names of Ataluren or Translarna and can be delivered orally. It has not yet been tried clinically for PXE patients.

One preclinical study examined the efficacy of PTC-124 in a "PXE" zebrafish morpholino model. Zhou et al. utilized HEK293 cells transfected with ABCC6 expression vectors harboring seven different PXE-nonsense mutations, including the most common stop codon mutation, p.R1141X, to evaluate whether PTC-124 could facilitate read-through of these variants [131]. Using immunostaining, they found that the amount of full-length protein synthesis was increased after 72 h of treatment with PTC-124 at a concentration of 5 µg/mL. To test the functionality of these potential full-length read-through ABCC6 proteins, they employed a zebrafish morpholino rescue system. As PTC-124 primarily replaces UAG PTC with tRNA corresponding to Trp, Cys, and Arg (here replaced by Gly), the authors of this study showed that these three possible read-through *ABCC6* variants rescued an *Abcc6a* morpholino-induced phenotype in zebrafish. Of note, in the *ABCC6* mutation database, a single missense variant affecting codon R1141 is reported as p.R1141Q and is classified as benign. It is therefore possible that substitutions at the R1141 site may not have negative impact. Despite these encouraging results, the study did not address calcification and there has been no follow-up study in mouse models of PXE.

One important argument for using PTC-124 would be restoration of the full physiological function of the entire ABCC6 → NPP1 → NT5E⊣TNAP pathway (Figures 1 and 3) as the *Enpp1*, *Nt5e* and *Alpl* genes are affected in the absence of *Abcc6* [11,18]. In addition, a large fraction of *ABCC6* disease-causing variants are nonsense, notably the R1141X variant, which is particularly prevalent in Caucasian PXE and GACI patients [169,170]. Another advantage favoring PTC-124 is its oral delivery and that only one allele needs to be targeted, as PXE and GACI are recessive diseases. However, these advantages are also limitations, as treatment would only be restricted to those carrying nonsense alleles.
