Most patients carry a mutation of one or more exons, e.g. exon 45 (Figure 1). As exon 45 contains 176 nucleotides (not divisible by 3) the deletion disrupts the open reading frame, resulting in the incorporation of aberrant amino acids and a premature truncation codon in exon 46. Since a deletion of both exons 45 and 46 covers 324 nucleotides (divisible by 3), this deletion maintains the open reading frame. To restore the open reading frame for patients with a deletion of exon 45, exon 46 is therefore hidden from the splicing machinery by a specific AON. This leads to the skipping of the targeted exon and an mRNA transcript for which the reading frame is restored. In addition to an exon 45 deletion (exon 46 skip), we have confirmed the therapeutic applicability of single exon skipping in cultured cells from patients with a deletion of exon 45-50 (exon 51 skip), exon 45-54 (exon 44 skip), exon 48-50 (exon 51 skip), exon 49-50 (exon 51 skip), exon 50 (exon 51 skip), exon 51-55 (exon 50 skip) and exon 52 (exon 51 or exon 53 skip).
Figure 1: Single exon skipping for deletions
For a small number of deletions, skipping a single exon is not sufficient to restore the open reading frame, e.g. a deletion of exon 46-50 (Figure 2). The total number of deleted nucleotides is 695 (not divisible by 3) and neither a deletion of 45-50 (871 nucleotides) or a deletion of 46-51 (928 nucleotides) is divisible by 3 and thus exon 45 skipping or exon 51 skipping will not restore the open reading frame.
However, a deletion of exon 45-51 involves 1106 nucleotides, which is divisible by 3. Therefore, to restore the open reading frame for this patient both exon 45 and exon 51 have to be skipped. This can be achieved by using a combination of AONs targeting exon 45 and AONs targeting exon 51. We have indeed confirmed the applicability of double exon skipping in cultured cells from a patient with a deletion of exon 46-50.
Figure 2: Double exon skipping for deletions