DG9-Conjugated Morpholino-Based Exon 51-Skipping Therapy for Duchenne Muscular Dystrophy.

Abstract

Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene that leads to the loss of the dystrophin protein. Exon-skipping therapy with phosphorodiamidate morpholino oligomers (PMOs) has been shown to restore the disrupted open-reading frame to produce a shortened yet functional dystrophin. The applications of this treatment, however, are limited in terms of its efficiency. A potential solution to this problem is to conjugate the PMOs to cell-penetrating peptides. DG9 is a novel peptide that has exhibited promising results in terms of its ability to enhance the cellular uptake of PMOs to increase exon-skipping efficiency in both the skeletal and heart muscles. In this chapter, we describe the systemic injection of DG9-conjugated PMOs to induce the skipping of exon 51 in an exon 52-deleted mouse model, hDMDdel52;mdx, which is a humanized dystrophic mouse model that mimics human DMD patients by having a human DMD gene integrated into them with a disrupted reading frame. As a result, this genetic makeup makes the model useful for testing the safety and efficacy of DG9-PMO as a potential treatment for DMD. We also describe the methodology to assess the efficacy and safety of the DG9-PMO treatment, including systematic DG9-PMO injections, RT-PCR, western blotting, histology, and functional tests.