Recessive Dystrophic Epidermolysis Bullosa (RDEB) is disabling genodermatosis, caused by COL7A1 gene mutations. However, different mutation types may have different treatment approaches. Diseases caused by mutations leading to shifting reading frames and premature termination codon (PTC) appearance could be mitigated by drugs causing stop codon skipping, such as gentamicin. This application is questionable owing to its cytotoxicity and nonspecific coverage of engaged transcripts. Nonsense-mediated mRNA decay (NMD) is the mechanism for translation-dependent elimination of aberrant transcripts with PTC. NMD inhibitor (NMDI14) disrupts the SMG7-UPF1 interaction and presumably does not interfere with other cell processes, contrary to PTC-skipping drugs. Here we design a reporter of NMD activity, compare the effects of different drugs with it, and explore the effect of NMDI14 in RDEB fibroblasts lines. To evaluate NMD activity in live cells we engineered the genetic construct encoded two fluorescent proteins, expressed independently. One of the proteins is encoded by the NMD insensitive template, and the mRNA of another is degraded due to splicing dependent NMD. We demonstrated the modalities of this reporter in the stable HeLa cell line, particularly after NMDI14 treatment in comparison to G418 and gentamicin. For therapeutic potential attest we studied the NMDI14 effect on four lines of RDEB patient fibroblasts, which carry mutations, leading to PTC, and one line of healthy donor fibroblasts. We inspected DDX6, as a P-bodies formation marker, and revealed its enhancement induced by NMDI14. The COL7A1 transcripts representation after NMDI14 treatment revealed the additional forms, which increased total COL7A1 transcript presence. This work provides a new vision on the therapy of nonsense-mutation diseases and points to the balance of short cytotoxicity NMDI application period and a lengthy presence of expressed long half-life proteins like collagen VII.

ESDR163 - Nonsense-mediated mRNA decay inhibitors perspectives for recessive dystrophic epidermolysis bullosa treatment

50th Annual Meeting of the European Society for Dermatological Research. VIRTUAL MEETING