Highly sensitive screening of antisense sequences for different types of DMD mutations in patients' urine-derived...

Exon skipping using short antisense oligonucleotides (AONs) is a promising treatment for Duchenne muscular dystrophy (DMD). Several exon-skipping drugs, including viltolarsen (NS-065/NCNP-01), have been approved worldwide. Immortalized human skeletal muscle cell lines, such as rhabdomyosarcoma cells, are frequently used to screen efficient oligonucleotide sequences. However, rhabdomyosarcoma cells do not recapitulate DMD pathophysiology as they express endogenous dystrophin. To overcome this limitation, we recently established a direct human somatic cell reprogramming technology and successfully developed a cellular skeletal muscle DMD model by using myogenic differentiation 1 (MYOD1)-transduced urine-derived cells (MYOD1-UDCs). Here, we compared in vitro drug screening systems in MYOD1-UDCs and rhabdomyosarcoma cells. We collected UDCs from patients with DMD amenable to exon 51 skipping, and obtained MYOD1-UDCs. We then compared the efficiency of exon 51 skipping induced by various morpholino-based AONs, including eteplirsen in differentiated MYOD1-UDCs (UDC-myotubes) and rhabdomyosarcoma cells. Exon skipping was induced more efficiently in UDC-myotubes than in rhabdomyosarcoma cells even at a low AON concentration (1 μM). Furthermore, exon 51 skipping efficiency was higher in UDC-myotubes with a deletion of exons 49-50 than in those with a deletion of exons 48-50, suggesting that the skipping efficiency may vary depending on the DMD mutation pattern. An essential finding of this study is that the sequence of eteplirsen consistently leads to much lower efficiency than other sequences. These findings underscore the importance of AON sequence optimization by our cellular system, which enables highly sensitive screening of exon skipping drugs that target different types of DMD mutations.