Antisense suppression of donor splice site mutations in the dystrophin gene transcript

Abstract

We describe two donor splice site mutations, affecting dystrophin exons 16 and 45 that led to Duchenne muscular dystrophy (DMD), through catastrophic inactivation of the mRNA. These gene lesions unexpectedly resulted in the retention of the downstream introns, thereby increasing the length of the dystrophin mRNA by 20.2 and 36 kb, respectively. Splice-switching antisense oligomers targeted to exon 16 excised this in-frame exon and the following intron from the patient dystrophin transcript very efficiently in vitro, thereby restoring the reading frame and allowing synthesis of near-normal levels of a putatively functional dystrophin isoform. In contrast, targeting splice-switching oligomers to exon 45 in patient cells promoted only modest levels of an out-of-frame dystrophin transcript after transfection at high oligomer concentrations, whereas dual targeting of exons 44 and 45 or 45 and 46 resulted in more efficient exon skipping, with concomitant removal of intron 45. The splice site mutations reported here appear highly amenable to antisense oligomer intervention. We suggest that other splice site mutations may need to be evaluated for oligomer interventions on a case-by-case basis.

Keywords: Duchenne muscular dystrophy; exon skipping; splice mutation; splice-switching oligomer.

Figure 1

Arrangement of dystrophin exons (A) 15–17, and (B) 43–47 indicating exon (upper case):intron (lower case italics) sequences, intron length (shown in italics) and splice site scores, indicated by block arrows. Both mutations (c.1992 + 1 g>t and c.6614 + 1 g>a) occur at the invariant first “g” of introns 16 and 45, respectively. Splice site scores were determined using the algorithm http://rulai.cshl.edu/new_alt_exon_db2/HTML/score.html, and the scores resulting from the mutations are shown in parentheses. (C) RT-PCR across dystrophin exons 12–17 from RNA prepared from two independent untreated DMD-16ss myogenic cell cultures (ut). A 100 bp ladder was used as a size standard.

Figure 2

RT-PCR across dystrophin exons 1–7 (A), and 69–75 (B) on four separate RNA preparations from patient DMD-16ss. RT-PCR on RNA prepared from DMD-16ss cells using a forward primer directed to exon 15 and a reverse primer targeting intron 16 (C), and a forward primer annealing near the end of intron 16 and a reverse primer targeting exon 20 (D). RNA from normal human myogenic cells was included for comparison. A 100 bp ladder was used as a size standard.

Figure 3

RT-PCR of RNA extracted from patient DMD-16ss cells transfected with a 2OMe modified antisense oligomer targeting exon 16, at concentrations indicated. The oligomer-induced amplicon missing exon 16 is 536 bp, and the smaller product (326 bp) is a revertant transcript missing exons 14–16 (reported previously Mitrpant et al. (2009)). The larger amplicon (∼700 bp) results from cryptic splicing, and is present in all samples.

Figure 4

Protein extracts from normal cells and oligomer-treated and untreated DMD-16ss cells (∼30 μg, standardized to myosin) were analyzed by western blotting as described previously (McClorey et al. 2006). The 2OMe AO was transfected as a cationic lipoplex at 400 nmol/L and PPMO-k was added directly at 2 μmol/L, and the cells were incubated for 7 days. Dystrophin expression was revealed by NCL-DYS2 (Novocastra Laboratories) and chemiluminescent detection with Western Breeze (Life Technologies). Myosin (lower band) is indicated.

Figure 5

RT-PCR across exons 1–7 (A), and 69–75 (B) of the normal and DMD-45ss dystrophin transcripts. (C) RT-PCR using a forward primer targeting exon 43 and a reverse primer annealing within intron 45 and (D) a forward primer annealing at the end of intron 45 with a reverse primer targeting exon 47.

Figure 6

Normal (left panel) and DMD-45ss myogenic cells (right panel) were transfected with 2OMe phosphorothioate oligomers targeting single exons 44 (A, F), 45 (B, G) or 46 (C, H) or the dual exon blocks, 44 and 45 (D, I), or 45 and 46 (E, J), at concentrations indicated. Nested amplification used a forward primer in exon 41 and a reverse primer in exon 47 to generate a full-length transcript product of 1032 bases. The sizes of exon-skipped products are indicated. A 100 bp size standard was used to estimate amplicon sizes.

Figure 7

RT-PCR from exon 43 to intron 45 to detect dystrophin transcripts retaining intron 45, in the presence and absence of oligomers targeting exons 44 and 45. The size of the amplicon expected from retention of intron 45 is indicated (1159 bp).