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

Abstract

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 UDCmyotubes 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.

Systemic administration of the antisense oligonucleotide NS‐089/NCNP‐02 for skipping of exon 44 in patients with Duchenne muscular dystrophy: Study protocol for a phase I/II clinical trial

“…However, only a few studies are conducted on MYOD-UDCs, it is desirable to use them in parallel with the well-characterized MYOD-fibroblasts model. 37 Very recently, we, for the first time, The modeling of truncated dystrophin protein lacking exons 44-45 or 45-46 predicts that both should result in comparable stable hybrid rod structure, suggesting that, in patients with exon 45 mutations, skipping of exon 44 or exon 46 should be excellent therapeutic strategies. 24 However, it is already known that the resulting protein is functional enough to maintain a mild BMD phenotype, which generally has mutations in the DMD gene that maintain the open reading frame, allowing the production of dystrophin proteins and thus are partially functional.…”

“…UDCs might be an ideal cellular model for neuromuscular diseases because they have the high proliferative ability and can be collected non‐invasively. However, only a few studies are conducted on MYOD‐UDCs, it is desirable to use them in parallel with the well‐characterized MYOD‐fibroblasts model 37 . Very recently, we, for the first time, evaluated the usefulness of MYOD‐UDCs from study subjects to evaluate the efficacy of NS‐089/NCNP‐02 in the DMD clinical trial.…”

Systemic administration of the antisense oligonucleotide NS-089/NCNP-02 for skipping of exon 44 in patients with Duchenne muscular dystrophy: study protocol for a phase I/II clinical trial

“…In this study, the in vitro assay, which includes the differentiation of urine-derived cells into myocytes, is based on the authors’ recently published technique to create a novel DMD muscle cell model [35]. This method could be used to screen different ASOs prior to commencing clinical trials [20, 36], potentially streamlining the therapeutic development process and reducing the time to availability of new treatment strategies. Recently, we have reported an efficient cellular skeletal muscle modeling in DMD using MYOD-UDCs obtained from DMD patients [35].…”

“…UDCs might be an ideal cellular model for neuromuscular diseases because they have the high proliferative ability and can be collected non-invasively. However, only a few studies are conducted on MYOD-UDCs, it is desirable to use them in parallel with the well-characterized MYOD-fibroblasts model [36]. Very recently, we, for the first time, evaluated the usefulness of MYOD-UDCs from study subjects to evaluate the efficacy of NS-089/NCNP-02 in the DMD clinical trial.…”