Dystrophin is a sarcolemma protein that acts as a link between the intracellular cytoskeleton and the extracellular matrix. Absence or deficiency of dystrophin, due to mutation in the DMD gene, cause progressive destruction of muscle and the consequent Duchenne muscular dystrophy (DMD) disease. One of the main therapeutic approaches for DMD is the restoration of internally shortened, but yet functional, dystrophin protein, by taking advantage of the splice-switching antisense oligonucleotides (AONs) approach. Several antisense oligonucleotides are now approved, including the phosphorodiamidate morpholino oligomers (PMOs) eteplirsen, casimersen, viltolarsen and golodirsen, this last one targeting exon 53 of the DMD gene.
To better understand the mechanism of action of golodirsen and the inter-patient variability shown in the clinical study 4053-101, we assessed the specific exon 53 PMO-induced skipping in patients’ cells. Fibroblasts, derived from the 25 patients enrolled in the study, were differentiated into myotubes by lentivirally-mediated MyoD and treated with golodirsen. For the first time, the effect of antisense treatment on the transcript 5’-3’ imbalance in human cells was evaluated by FluiDMD cards, showing a modest but significant transcript imbalance restoration in 82% of treated patients. To understand the intracellular dynamics and subcellular localization of the skipped products compared to transcripts that had not been therapeutically skipped, we used specific BaseScope probes recognising transcripts with or without the DMD exon 53. Using Wes analysis, we were able to evaluate the restoration of dystrophin protein and its different molecular weight, in treated patient cells compared to healthy donors.
Finally, skipping efficiency and transcript imbalance restoration were correlated with specific patient's genotypes, after detection of the deletion breakpoint coordinates.
Our study is providing additional information on the dynamics of DMD mRNA in patients and how this is affected by AON treatment.
This work was funded by Sarepta Therapeutics, Inc.