Significance Duchenne muscular dystrophy (DMD) is a fatal disorder of progressive body-wide muscle weakness, considered the most common muscular dystrophy worldwide. Most patients have out-of-frame deletions in the DMD gene, leading to dystrophin absence in muscle. There is no cure for DMD, but exon skipping is emerging as a potential therapy that uses antisense oligonucleotides to convert out-of-frame to in-frame mutations, enabling the production of truncated, partially functional dystrophin. Currently approved exon skipping therapies, however, have limited applicability and efficacy. Here, we developed a more economical approach to skip DMD exons 45 to 55 (a strategy that could treat nearly half of all DMD patients) and identified DG9 peptide conjugation as a powerful way to improve exon skipping efficiencies in vivo.
Exon skipping using antisense oligonucleotides (ASOs) has recently proven to be a powerful tool for mRNA splicing modulation. Several exon-skipping ASOs have been approved to treat genetic diseases worldwide. However, a significant challenge is the difficulty in selecting an optimal sequence for exon skipping. The efficacy of ASOs is often unpredictable, because of the numerous factors involved in exon skipping. To address this gap, we have developed a computational method using machine-learning algorithms that factors in many parameters as well as experimental data to design highly effective ASOs for exon skipping. eSkip-Finder (https://eskip-finder.org) is the first web-based resource for helping researchers identify effective exon skipping ASOs. eSkip-Finder features two sections: (i) a predictor of the exon skipping efficacy of novel ASOs and (ii) a database of exon skipping ASOs. The predictor facilitates rapid analysis of a given set of exon/intron sequences and ASO lengths to identify effective ASOs for exon skipping based on a machine learning model trained by experimental data. We confirmed that predictions correlated well with in vitro skipping efficacy of sequences that were not included in the training data. The database enables users to search for ASOs using queries such as gene name, species, and exon number.
Antisense oligonucleotide (AO)-mediated therapy is a promising strategy to treat several neurological diseases including spinal muscular atrophy (SMA). However, limited delivery to the central nervous system (CNS) with AOs administered intravenously or subcutaneously is a major challenge. Here we demonstrate a single subcutaneous administration of cell-penetrating peptide DG9 conjugated to an AO called phosphorodiamidate morpholino oligomers (PMOs) reaches the CNS and significantly prolonged the median survival compared to unconjugated PMO and R6G-PMO in a severe SMA mouse model. Treated mice exhibited significantly higher expression of full-length SMN2 expression (FL-SMN2) in both the CNS and systemic tissues compared to non-treated and unmodified AO-treated mice. The treatment ameliorated the atrophic musculature and improved breathing function accompanied by improved muscle strength and innervation at the neuromuscular junction with no signs of apparent toxicity. We also demonstrated DG9-conjugated PMO localizes in nuclei in the spinal cord and brain after subcutaneous injections. Our data identify DG9 peptide conjugation as a powerful way to improve the efficacy of AO-mediated splice modulation. Finally, DG9-PMO is a promising therapeutic option to treat SMA and other neurological diseases, overcoming the necessity for intrathecal injections and treating body-wide tissues without apparent toxicity.
Dystrophin is a 427 kDa protein that stabilizes muscle cell membranes through interactions with the cytoskeleton and various membrane-associated proteins. Loss of dystrophin as in Duchenne muscular dystrophy (DMD) causes progressive skeletal muscle weakness and cardiac dysfunction. Multiple promoters along the dystrophin gene (DMD) give rise to a number of shorter isoforms. Of interest is Dp71, a 71 kDa isoform implicated in DMD pathology by various animal and patient studies. Strong evidence supporting such a role for Dp71, however, is lacking. Here, we use del52;WT mice to understand how Dp71 overexpression affects skeletal and cardiac muscle phenotypes. Apart from the mouse Dmd gene, del52;WT mice are heterozygous for a full-length, exon 52-deleted human DMD transgene expected to only permit Dp71 expression in muscle. Thus, del52;WT mice overexpress Dp71 through both the human and murine dystrophin genes. We observed elevated Dp71 protein in del52;WT mice, significantly higher than wild-type in the heart but not the tibialis anterior. Moreover, del52;WT mice had generally normal skeletal muscle but impaired cardiac function, exhibiting significant systolic dysfunction as early as 3 months. No histological abnormalities were found in the tibialis anterior and heart. Our results suggest that Dp71 overexpression may have more detrimental effects on the heart than on skeletal muscles, providing insight into the role of Dp71 in DMD pathogenesis.
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