VP.58 Golodirsen induced DMD transcripts localization and dystrophin production in MyoD-converted fibroblasts from 4053-101 clinical trial patients

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.