P29 DMD transcript imbalance and nuclear trafficking evaluation in muscle biopsies from baseline and golodirsen treated 4053-101 clinical trial patients

Duchenne muscular dystrophy (DMD) is an X-linked, rare, neuromuscular disease caused by mutations in the DMD gene affecting its reading frame. This results in a substantial reduction or absence of dystrophin protein and a non-uniform expression of the DMD transcript throughout its 14-kb length, the so-called transcript imbalance. Recent evidence has also found an impairment in the sub-cellular localization of the DMD transcript in mdx mice, demonstrating nuclear retention, compared to wild type. Golodirsen, a phosphorodiamidate morpholino oligomer (PMO), is a promising therapeutic agent designed to skip exon 53 of the DMD transcript, restoring the reading frame and producing a truncated yet functional dystrophin protein. This study aims to confirm the restorative role of golodirsen on transcript imbalance and to assess if the intracellular trafficking of the skipped DMD mRNA transcript differs from the mutant one, in human. Using FluiDMD cards, we evaluated the transcript imbalance ratio in the skeletal muscle biopsies from 25 DMD patients recruited into the clinical study 4053-101, at baseline and after 48 weeks of treatment (W48). In eight of these biopsies were also investigated by in-situ RNA hybridization using the RNA-scope approach to visualise the mutant and skipped transcripts subcellular localization. Our results demonstrate a slight but significant imbalance restoration in 40% of the W48 skeletal muscle biopsy samples. Comparative analysis with our previous results on MyoD-converted fibroblasts derived from the same patients demonstrates that the transcript imbalance was restored more efficiently in the MyoD-converted fibroblasts, in which golodirsen corrected imbalance in 80% of samples. Thos could be related to the more efficient exon skipping achieved in the MyoD converted fibroblast compared to the in-vivo delivery of golodirsen or to the differences between the in vitro fibroblast-derived myotubes and muscle fibers. Increasing the DMD transcript knowledge could elucidate this aspect and might help in better understanding the inter-patient response variability.