Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic disorders. The DMD gene encodes the protein Dystrophin that supports the muscle membrane. Out-of-frame mutations in this gene lead to the loss of dystrophin protein which predisposes the muscle fibres to damage. While there are some relieving methods, a cure remains elusive. Exon skipping is a technique that typically employs antisense oligonucleotides such as phosphorodiamidate morpholino oligomers (PMOs) to skip over the frame-disrupting part of the DMD gene and restore the reading frame. Although 4 PMOs have been approved for treating DMD, none is targeted for exon 44. Moreover, PMOs show limited efficacy in the heart. Here, we developed a novel peptide-conjugated PMO (PPMO) to skip exon 44 and restore dystrophin production in skeletal and cardiac muscles.
Based on our in silico analysis, we evaluated the activity of multiple PMOs on patient muscle cell lines that have an exon 45 deletion in the DMD gene. From this in vitro data, the most efficient PMO at skipping exon 44 and restoring exons 44-45 skipped in-frame mRNA was then conjugated to a cell-penetrating peptide called “DG9” and was further tested in humanized DMD model mice carrying the human DMD gene with an out-of-frame exon 45 deletion. Molecular analysis showed that systemic treatment with DG9-PMO restored 17% and 40% dystrophin compared to healthy control mice in the tibialis anterior and cardiac muscles, respectively, with no overt toxicity. Functional analysis revealed that the treatment improved muscle function including grip strength and endurance. Moreover, the treatment was able to provide significant cardio-protection against β-isoproterenol-induced cardiac damage.
In conclusion, our identified DG9-PMO improved muscle function and restored bodywide dystrophin production accompanied by the functional improvement and cardiac protection. This study lays the foundation for a promising PPMO to advance into clinical trials.