Targeted exon skipping to correct exon duplications in the dystrophin gene

Abstract

Duchenne muscular dystrophy is a severe muscle-wasting disease caused by mutations in the dystrophin gene that ablate functional protein expression. Although exonic deletions are the most common Duchenne muscular dystrophy lesion, duplications account for 10-15% of reported disease-causing mutations, and exon 2 is the most commonly duplicated exon. Here, we describe the in vitro evaluation of phosphorodiamidate morpholino oligomers coupled to a cell-penetrating peptide and 2'-O-methyl phosphorothioate oligonucleotides, using three distinct strategies to reframe the dystrophin transcript in patient cells carrying an exon 2 duplication. Differences in exon-skipping efficiencies in vitro were observed between oligomer analogues of the same sequence, with the phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide proving the most effective. Differences in exon 2 excision efficiency between normal and exon 2 duplication cells, were apparent, indicating that exon context influences oligomer-induced splice switching. Skipping of a single copy of exon 2 was induced in the cells carrying an exon 2 duplication, the simplest strategy to restore the reading frame and generate a normal dystrophin transcript. In contrast, multiexon skipping of exons 2-7 to generate a Becker muscular dystrophy-like dystrophin transcript was more challenging and could only be induced efficiently with the phosphorodiamidate morpholino oligomer chemistry.

Figures

Figure 1

AO strategies to restore dystrophin expression in the presence of a frame-shifting exon 2 duplication. (a) Possible outcomes resulting from targeting of exon 2 only include single exon skipping (and a resultant in-frame transcript) or excising both copies of exon 2 and relying on reinitiation of translation from exon 3 or 6. (b) Induction of multiple exon skipping targeting exons 2–7 to restore the reading frame. AO, antisense oligomer.

Figure 2

Schematic showing predictedSR proteins binding motifs in dystrophin exon 2, with AO annealing sites indicated. Lower case letters indicates intronic sequences, while the exon is shown in upper case. The colored bars represent different motifs for predicted SR binding proteins. The heights of the bars indicate predicted binding strength. Red: SRSF1; purple: SRSF1 (IgM-BRCA1); green: SRSF5; yellow: SRSF6. AO, antisense oligomer; SR, specific serine/arginine rich; SRSF, specific serine/arginine rich splicing factor.

Figure 3

AO-induced excision of dystrophin exon 2 from normal human myogenic cells and patient dystrophin myogenic cells with a duplication of exon 2, using oligomers of the 2OMe and PPMO chemistries. Myogenic cells were either transfected with 2OMe AO:cationic lipoplexes at the concentrations indicated and incubated for 24 hours, or transfected with PPMOs and incubated for 96 hours. Total RNA was extracted and nested reverse-transcriptase PCR was undertaken across exons 1–4. The normal, full length, and exon 2-deleted products are 389, 451, and 327 bp, respectively. (a) Normal human myogenic cells transfected with 2OMe; (b) exon 2 duplication cells with 2OMe; (c) normal human myogenic cells with PPMO; (d) exon 2 duplication cells with PPMO; (e) sequence chromatogram showing exon 1 being spliced to exon 3; (f) schematic indicating exonic combinations and predicted amplicon size. AO, antisense oligomer; PPMO, phosphorodiamidate morpholino oligomer, coupled to a cell-penetrating peptide; UT: amplicon from untreated exon 2 duplication cells; +ve: amplicon from untreated normal cells; −ve: no template PCR negative control; 2OMe, 2′O methyl phosphorothioate oligomers.

Figure 4

Induced removal of dystrophin exons 2–7 from normal human myogenic and exon 2 duplication myogenic cells using 2OMe AO and PPMO cocktails. Primary human and dystrophic myogenic cell cultures were transfected with either 2OMeAO:cationic lipoplexes at the concentrations indicated and incubated for 24 hours or transfected with PPMO conjugates targeting dystrophin exons 2–7 and left for 96 hours. Total RNA was extracted and nested RT-PCR was undertaken across exons 1–10. The normal, full length, and exon 2–7 deletion transcripts are represented by products of 1,157, 1,219, and 539 bp, respectively. (a) Normal myogenic cells treated with the 2OMe cocktail. (b) Exon 2 duplication cells treated with the 2OMe cocktail. (c) Normal myogenic cells treated with the PPMO cocktail. (d) Exon 2 duplication cells treated with the PPMO cocktail. (e) Sequence chromatogram confirming identity of 539 and 210 bp amplicons. Mixed sequencing peaks are generated downstream of exon 1 due to templates representing transcripts missing exons 2–7 and 2–9. (f) Schematic indicating induced exon-skipping product sizes. AO, antisense oligomer; PPMO, phosphorodiamidate morpholino oligomer, coupled to a cell-penetrating peptide; UT: amplicon from untreated exon 2 duplication cells; +ve: amplicon from untreated normal cells; −ve: no template PCR negative control; 2OMe, 2′O methyl phosphorothioate oligomers.