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Simona Torriano, Nejla Erkilic, David Baux, Valerie de Luca, Nicholas Cereso, Mariya Moosajee, Christian P Hamel, Anne-Francoise Roux, Vasiliki Kalatzis; COMPREHENSIVE ANALYSIS OF PTC-124 THERAPY FOR CHOROIDEREMIA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):622.
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© ARVO (1962-2015); The Authors (2016-present)
Choroideremia (CHM) is a X-linked retinal dystrophy. The causative gene, CHM, encodes REP1, Rab escort protein-1. REP1 binds unprenylated Rab GTPases and presents them to the Rab generanylgeranyl transferase for prenylation, which renders them functional. Approximately 30% of all CHM patients carry a nonsense mutation. In these patients, REP1 is unstable, but the persistence of mRNA provides a template to test the efficiency of read-through drugs, such as PTC-124.
We assayed the efficiency of PTC-124 in fibroblasts from CHM patients carrying different nonsense codons (UGA, UAA, UAG) and in patient-specific induced pluripotent stem cell (iPSc)-derived retinal pigment epithelium (RPE). We evaluated the restoration of function by in vitro prenylation.
In a few experiments, PTC-124 rescued prenylation by 50% compared to untreated cells, but, surprisingly, in the majority of the experiments, treatment had a negative effect on the prenylation process. REP1 was never detected post-treatment. Furthermore, to exclude a detection threshold of our readout, we generated a CHM-GFP fusion protein plasmid, which was mutated to introduce two of the three patient mutations. Transfected COS7 cells were treated with PTC-124 and FACS analysis of GFP expression did not detect a significant increase post-treatment. Given that the replacement of the premature stop codon by an amino acid is random, we hypothesised that if the mutation is situated in a highly conserved position or in an important functional domain, PTC-124 efficacy could be decreased. We therefore performed in silico predictions to assay the effect of such changes on REP1 stability. These predictions show that the mutations present in our patients concern highly conserved amino acid residues and changes are predicted to have deleterious effects on REP1, in accordance with what we observed in our human cellular models.
Although our results did not allow us to confirm the effectiveness of this drug, they do not suggest that PTC-124 is not effective in general. We are highlighting aspects that should be considered when using these kinds of pharmacological molecules. Many unexpected factors must be evaluated for any therapy; here we take into account some of the genetic factors that could affect a PTC-124 therapy, with particular attention to Choroideremia.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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