Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping.

RNA processing in skeletal muscle biology and disease

“…by a reading frame mutation in the DMD gene which is 79 exons long and encodes the dystrophin protein which in muscles links actin with the extracellular matrix [142][143][144] (Figure 8(a)). Mis-splicing of some exons within the DMD gene has also been implicated in DMD; however, splicing variants contribute to less than 7% of all disease-causing variants.…”

“…In 2016, the FDA approved the ASO known as Eteplirsen to promote the skipping of exon 51 in DMD pre-mRNA [143]. Skipping of exon 51 restores the reading frame and thus produces a partially functional DMD protein [143,144] (Figure 8(b)). Since Eteplirsen is applicable to approximately 14% of all DMD cases [143], other ASO technologies are currently in clinical trials [145].…”

Novel PYGL mutations in Chinese children leading to glycogen storage disease type VI: two case reports

“…For example, antisense oligonucleotide-mediated exon skipping is an emerging therapy for Duchenne muscular dystrophy patients. Exon skipping can restore the reading frame by removing the mutant exon and/or its flanking exon(s) from the pre- mRNA, leading to the expression of truncated but functional proteins [14]. The effect of this gross deletion on the liver phosphorylase requires further functional assays using either RNA or mutant protein analysis.…”

Effectiveness of pharmacological treatments in Duchenne muscular dystrophy: a protocol for a systematic review and meta-analysis

“…Ataluren and gentamicin have a different pharmacological mechanism of action than that of eteplirsen and drisapersen. Therefore, they are targeted to different subgroups of patients and they cannot be exchanged with one another, and only the efficacy of drugs with the same target population can be compared, that is, gentamicin versus ataluren and eteplirsen versus drisapersen In particular, this last case can shed light on why eteplirsen is approved by the FDA,12 unlike drisapersen,28 and whether that authorisation is justified. In contrast, the comparison of drugs with different mechanisms of action will indicate which subgroup of patients can potentially benefit more.…”