Enhanced exon skipping in the 4CV dystrophic mouse model of muscular dystrophy through refined oligomer design

Antisense oligomer (AO) induced dual dystrophin exon skipping was studied in the B6Ros.Cg-Dmdmdx-4Cv/J (4CV) mouse, an established mouse dystrophinopathy model carrying a nonsense mutation in DMD exon 53. We previously reported by-passing this mutation by AO-induced exclusion of exons 52 and 53. However, differences in the efficiency of removal of exons 52 and 53 led to a mixture of transcripts, with some missing only exon 53 and others missing both exons. We have developed different AO cocktails and report a more effective combination of AOs that induced almost exclusive expression of the desired transcript missing both exons, first in vitro and then in vivo. This combination led to substantial dystrophin restoration and improved muscle functionality. A panel of AOs with phosphorothioate backbone (2OMe) were designed and evaluated as the AO cocktails to induce exons 52 and 53 removal from the dystrophin gene transcript expressed in cultured 4CV myoblasts. Exon skipping efficiency of the corresponding sequences prepared as phosphorodiamidate morpholino oligomers (PMOs) cocktails was evaluated in vitro and in vivo at RNA and protein level. Both intramuscular and intraperitoneal administration of an AO-cocktail preferentially induced the in-frame dystrophin transcript missing both exons and partially restored dystrophin protein expression in vivo. The oligomer cocktail also restored assembly of the dystrophin-associated protein complex (DAPC), as demonstrated by expression and localisation of nNOS and β-dystroglycan. PMO treatment also led to partial restoration of diaphragm function in 4CV mice.