Splice switching antisense oligomers

An antisense oligomer, composed of 2’-O-methyl modified bases on a phosphorothioate backbone, was synthesized on an Expedite 8909 Nucleic Acid synthesizer using reagents from Azco Biotech (Ca, USA). Designed to induce DMD exon 6 skipping, the AO (5’-UAC GAG UUG AUU GUC GGA CCC AG-3’) annealed to bases 69–91 in exon 6, and had been shown to induce specific exon 6 skipping [21].

A panel of splice switching AOs was developed to excise mouse dystrophin exon 5, targeting similar coordinates used to excise human dystrophin exons 5 (Table 1). The 2’-O-methyl modified oligomer 5’ UAU GAU UUC CAU CCA CUA UGU CAG UGC UUC 3’ (Table 1, underlined) annealing to bases 20–49 in mouse dystrophin exon 5, was identified to efficiently remove the target exon 5 in vitro. This sequence was synthesized as a phosphorodiamidate morpholino oligomer coupled to the cell penetrating peptide k [22] (PPMO) for in vivo experiments.

The underlined sequence (M5A(+20+49) was synthesised as a phosphorodiamidate morpholino oligomer coupled to the cell penetrating peptide k [22].