DMD is a serious muscle wasting condition for which there is no treatment. The application of splice switching oligomers to remove or correct frame-shifting mutations that would otherwise lead to premature termination of translation has progressed from a concept to clinical trials. Proof-of-concept has been demonstrated in animal models and recently in DMD patients, with completion of Phase I intramuscular studies and Phase I/IIa systemic studies with two different oligonucleotide analogues [5–8]
. The DMD mutation profile and incidence will determine exon targets for subsequent splice switching clinical trials, and these studies must focus on single exon removal, due to the number of responsive patients and cost of safety and toxicology validation required for each new oligomer 
. Dual exon skipping to restore dystrophin expression has been demonstrated in animal models [3,12,31,32]
and is technically feasible, but will be further from the clinic. The targeting of two or more exons in human dystrophin has been reported by others using AO cocktails [22,29,33]
or AOs joined by a linker.
DMD is regarded as an orphan disease, with an estimated 420,000 affected individuals worldwide 
. However, when further classifying amenable DMD mutations into subclasses, we suggest that exon skipping should be regarded as a personalised genetic treatment. As such, any strategies to implement exon skipping should be considered on a case-by-case basis, and it is important that the simplest and most effective exon skipping strategy for each mutation be carefully considered. The factors that will determine the most suitable strategy and AO sequence include the following: Which AOs are the most efficient at excising the target exon(s) in vitro
? Which strategy will produce the most functional truncated dystrophin isoform, determined by BMD patient data and transient inducible mouse model 
? What are the potential off-target effects of the AO treatment, including effects related to AO chemistry and sequence dependant effects? How many other patients could potentially be treated by skipping the combination of exons 
This study reports DMD mutations that can be addressed by multiple strategies to restore the reading frame and facilitate translation of a BMD-like dystrophin isoform. This includes intra-exonic mutations in exons 51 and 20. In these cases, the mutated exon must be excised with either flanking exon. The mutation in exon 51, was marginally more efficiently addressed, at least in vitro
, by skipping of exon 51 + 52. This is in contrast to the result in normal myogenic cells where exons 50 + 51 were more efficiently removed. ESE analysis did not show any alterations to ESEs as a result of the mutation, yet this is not conclusive evidence that the exon 51 disease-causing variant does not influence splicing. We previously demonstrated that a nonsense mutation in dystrophin exon 17 did not alter the efficiency of oligomers targeted to excise exon 17, but did influence exon 18 skipping 
. This suggests subtle cross communication between exons during splicing, and further emphasises the need for empirical AO testing in patient-derived cells. In terms of the effect on the functionality of the resulting dystrophin isoform, it is not yet clear if dystrophin lacking exon 50&51, and therefore the whole of hinge 3 will be more functional than dystrophin lacking exons 51&52 where part of hinge 3 is retained. It has been reported that in-frame dystrophin deletions involving hinge 3 have a milder phenotype compared with other shorter deletions that do not involve that domain 
In cells from the patients with small intra-exonic deletions in exon, exons 20&21 were consistently removed more efficiently than exons 19&20. This is in agreement with the results obtained in normal myogenic cells. Both these mutations did cause small changes in the predicted ESE sites, but this does not appear to have affected the efficiency of the AO cocktails. Again, the effect on the functionality of the resultant dystrophin must be considered. In an AO induced transient mouse model, exons 19&20 could be removed from 100% of transcripts in the mouse diaphragm, with no detectable detrimental effects on the muscle phenotype 
. The Leiden DMD database contains 2 patients lacking exons 19&20 [36,37]
, both are classified as DMD however the mutation screening was conducted using multiplex PCR and may not be fully informative 
. One patient with a deletion of exons 20&21 is classified as BMD (DMD mutation database on http://www.dmd.nl
), although detailed clinical data is not publicly available for these patients.
We also report on single base changes that alter splicing at particular exon boundaries and present unique opportunities for simpler, alternative exon skipping strategies. In these cases, additional factors should be considered, other than which is the most efficient strategy and which results in the most functional dystrophin isoform. We have demonstrated that for two splice site mutations an opportunity exists to restore the reading frame by excision of a single exon, rather than the two exons suggest by conventional exon skipping strategies. For the exon 22 acceptor splice site mutation, the results demonstrate that skipping both exons 21&22 is inefficient in the patient’s cells, while skipping exon 21 alone is a much simpler strategy and is more efficient in vitro
in patient cells. Some large in-frame DMD
deletions are associated with a mild phenotype, however this is greatly dependent upon the location of the DMD
. In general, the loss of a smaller region of the dystrophin protein should confer greater functionality, and therefore a milder phenotype. In addition to the loss of exon 21 sequence, this strategy results in an isoleucine to valine amino acid substitution. Both amino acids possess large, non-polar, strongly hydrophobic side chains; hence the substitution could be predicted to have a minimal effect on the structure and function of the protein.
In normal myoblasts, skipping exon 20&21 or exon 21&22 was comparable, with the 20&21 cocktail proving marginally more efficient. In MyoD converted fibroblasts from the patient carrying the exon 21 splice site mutation, exon 20&21 were effectively removed by an AO cocktail. Exon 20 was also effectively excised on its own. In this case the inserted bases at the beginning of exon 21 (AG) are identical to the last two bases of exon 20, so excision of exon 20 does not alter the remaining amino acid sequence. Unexpectedly, it appears that the mutation facilitates skipping of exon 21 when exon 20 only is targeted, possibly because the exon 21 acceptor splice site is compromised. However, as both these transcripts are in-frame and allow dystrophin translation, targeting exon 20 only remains a viable option for this patient, despite this unexpected effect on exon selection.
We propose a set of guidelines to be followed when dealing with splice site mutations that may be amenable to splice manipulation. The “minimalist” exon skipping strategies will be more applicable to mutations that occur near exon boundaries, and to minimise the possibility of encountering in-frame stop codons, will be very dependent on the precise position and nature of the mutation. Therefore, careful analysis of the effect of the mutation on splicing of the exon is required and it must also be determined if the mutation affects the AO binding sites. Many exons are most efficiently removed by an AO targeted near the 3′ acceptor splice site and mutations in this region would therefore disrupt AO binding, and it may be necessary to use sub-optimal AOs that avoid the mutation, or design additional sequences specific to the mutation. Once these considerations have been taken into account, the other factors that impact upon the optimal strategy; efficient removal of the target exon, functionality of the resultant dystrophin, potential off-target effects, applicability to other DMD patients and cost-benefit analysis of the alternative strategies must be considered, before the optimal strategy is selected.