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.

Antisense-mediated exon skipping, which can restore the reading frame, is a most promising therapeutic approach for Duchenne muscular dystrophy. Remaining challenges include the limited applicability to patients and unclear function of truncated dystrophin proteins. Multiexon skipping targeting exons 45–55 at the mutation hotspot of the dystrophin gene could overcome both of these challenges. Previously, we described the feasibility of exons 45–55 skipping with a cocktail of Vivo-Morpholinos in vivo; however, the long-term efficacy and safety of Vivo-Morpholinos remains to be determined. In this study, we examined the efficacy and toxicity of exons 45–55 skipping by intravenous injections of 6 mg/kg 10-Vivo-Morpholino cocktail (0.6 mg/kg each vPMO) every 2 weeks for 18 weeks to dystrophic exon-52 knockout (mdx52) mice. Systemic skipping of the entire exons 45–55 region was induced, and the Western blot analysis exhibited the restoration of 5–27% of normal levels of dystrophin protein in skeletal muscles, accompanied by improvements in histopathology and muscle strength. No obvious immune response and renal and hepatic toxicity were detected at the end-point of the treatment. We demonstrate our new regimen with the 10-Vivo-Morpholino cocktail is effective and safe for long-term repeated systemic administration in the dystrophic mouse model.

The main approach of AON therapy in DMD and SMA is to rescue ('knock up' or increase) target proteins through exon skipping or exon inclusion; conversely, most conventional antisense drugs are designed to knock down (inhibit) the target. Encouraging preclinical data using this 'knock up' approach are also reported to rescue dysferlinopathies, including limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, distal myopathy with anterior tibial onset and Fukuyama congenital muscular dystrophy.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder affecting motor neurons, and is currently the most frequent genetic cause of infant mortality. SMA is caused by a loss-of-function mutation in the survival motor neuron 1 (SMN1) gene. SMN2 is an SMN1 paralogue, but cannot compensate for the loss of SMN1 since exon 7 in SMN2 mRNA is excluded (spliced out) due to a single C-to-T nucleotide transition in the exon 7. One of the most promising strategies to treat SMA is antisense oligonucleotide (AON)-mediated therapy. AONs are utilized to block intronic splicing silencer number 1 (ISS-N1) on intron 7 of SMN2, which causes exon 7 inclusion of the mRNA and the recovery of the expression of functional SMN protein from the endogenous SMN2 gene. We developed novel locked nucleic acid (LNA)-based antisense oligonucleotides (LNA/DNA mixmers), which efficiently induce exon 7 inclusion in SMN2 and restore the SMN protein production in SMA patient fibroblasts. The mixmers are highly specific to the targeted sequence, and showed significantly higher efficacy than an all-LNA oligonucleotide with the equivalent sequence. These data suggest that use of LNA/DNA mixmer-based AONs may be an attractive therapeutic strategy to treat SMA.

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Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide (AON)-mediated therapy. While SMN1 mutations are responsible for the disease, AONs targeting intronic splice silencer (ISS) sites in SMN2, including FDA-approved nusinersen, have been shown to restore SMN expression and ameliorate the symptoms. Currently, many studies involving AON therapy for SMA focus on investigating novel AON chemistries targeting SMN2 that may be more effective and less toxic than nusinersen. Here, we describe a protocol for in vitro evaluation of exon inclusion using lipotransfection of AONs followed by reverse transcription polymerase chain reaction (RT-PCR), quantitative polymerase chain reaction (qPCR), and Western blotting. This method can be employed for various types of AON chemistries. Using this method, we demonstrate that AONs composed of alternating locked nucleic acids (LNAs) and DNA nucleotides (LNA/DNA mixmers) lead to efficient SMN2 exon inclusion and restoration of SMN protein at a very low concentration, and therefore, LNA/DNA mixmer-based antisense oligonucleotides may be an attractive therapeutic strategy to treat splicing defects caused by genetic diseases. The in vitro evaluation method described here is fast, easy, and sensitive enough for the testing of various novel AONs.

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