Exon skipping for point mutations
Small mutations can directly lead to premature stop codons (nonsense mutation) or disrupt the reading frame (intra exonic deletion or duplication of a number of nucleotides that is not divisible by 3). Either way, if the mutation is present in an in-frame exon, such as exon 49 that contains 102 nucleotides (divisible by 3), the mutation can be bypassed by skipping said exon (Figure 1). This has been confirmed in cultured cells from a patient with a nonsense mutation in exon 49.
If the small mutation is present in an exon that contains a number of nucleotides not divisible by 3 (e.g. exon 43 contains 173 nucleotides), skipping of the mutated exon will bypass mutation, but cause a disruption of the reading frame at the same time (i.e. exon 42 and exon 44 do not fit). However, a deletion of exon 43 and exon 44 is in-frame (321 nucleotides, divisible by 3). Thus by inducing the combined skipping of both exon 43 and exon 44 the mutation can be bypassed, while the reading frame is maintained (Figure 2). Treating cultured cells from a patient with the deletion of a single nucleotide in exon 43 with AONs targeting exon 43 and AONs targeting exon 44 indeed resulted in the generation of dystrophin.