**4.1 Single exon skipping**

First proofs-of-principle for the feasibility of restoring the reading frame by exon skipping have been shown *in vitro* in cultured primary human myoblasts, derived from DMD patients and in *mdx*-cell cultures.

In the early nineties, a DMD patient (named "DMD Kobe") was identified carrying a deletion of 52 base pairs within exon 19, which led to the skipping of the whole exon. The authors hypothesised that this sequence might be important for splicing. An AON targeting part of this exon induced exon skipping in human control lymphoblastoid cells (Pramono et al., 1996; Takeshima et al., 1995). In cells derived from a patient with a deletion of exon 20, PS AONs (able to activate RNase H) against the aforementioned sequence, resulted in exon 19 skipping and the restoration of dystrophin in ~20% of treated cells (Takeshima et al., 2001). By that time, exon skipping with 2OMePS AONs (unable to activate RNase H), had also been explored. In 2 patients with an out-of-frame exon 45 deletion treatment with AONs resulted in exon 46 skipping, which should restore the open reading frame. Exon skipping levels were ~15%, which restored the synthesis of functional dystrophin in more than 75% of the cells (van Deutekom et al., 2001). Subsequently, the skipping of different exons has been reported for patient-derived cells with other deletions, point mutations and duplications. For an overview see Aartsma-Rus, *RNA Biology* 2010 (Aartsma-Rus, 2010). Restoration of dystrophin synthesis was detectable at the membrane and the (at least partial) functionality of these BMD-like proteins was suggested by the reformation of the dystrophin-glycoprotein complex, shown by increased membrane expression of DGCassociated proteins. Another interesting finding were the higher exon skipping levels observed in these patient cell lines, than previously seen in control cell lines. This can be explained by nonsense-mediated decay (NMD) of the original out-of-frame transcripts, which are less stable than the newly formed in-frame transcripts (Aartsma-Rus et al., 2003).

#### **4.2 Double and multiple exon skipping**

In theory, skipping of a single exon would be beneficial for approximately 64% of the known mutations in DMD patients. However, there still is a large population which requires the skipping of 2 or more exons for reading frame restoration (Aartsma-Rus et al., 2009a). The theoretic applicability of exon skipping could be extended to 79% by double exon skipping and around 90% of patients by multiple exon skipping. Feasibility of double exon skipping was first shown in 2 different patient cell lines. One patient had a nonsense mutation in exon 43, for which dystrophin synthesis could be restored by skipping of exon 43 and 44. The second, carrying an exon 46-50 deletion, was successfully treated with a combination of AONs against exon 45 and 51. Dystrophin synthesis was found in 70% of the myotubes, which is almost as high as after single exon skipping (75-80%) (Aartsma-Rus et al., 2004a). Subsequently, successful double exon skipping has been proved by other groups (reviewed in Aartsma-Rus, *RNA Biology* 2010 (Aartsma-Rus, 2010)). The dog model for DMD needs double skipping to bypass the mutation and cells derived from these dogs have been used to show double exon skipping *in vitro* (see below) (McClorey et al., 2006).

A surprising finding in control myotubes was that combinational treatment with 45AON and 51AON caused the skipping of the entire stretch of exons from 45 through 51. This

First proofs-of-principle for the feasibility of restoring the reading frame by exon skipping have been shown *in vitro* in cultured primary human myoblasts, derived from DMD

In the early nineties, a DMD patient (named "DMD Kobe") was identified carrying a deletion of 52 base pairs within exon 19, which led to the skipping of the whole exon. The authors hypothesised that this sequence might be important for splicing. An AON targeting part of this exon induced exon skipping in human control lymphoblastoid cells (Pramono et al., 1996; Takeshima et al., 1995). In cells derived from a patient with a deletion of exon 20, PS AONs (able to activate RNase H) against the aforementioned sequence, resulted in exon 19 skipping and the restoration of dystrophin in ~20% of treated cells (Takeshima et al., 2001). By that time, exon skipping with 2OMePS AONs (unable to activate RNase H), had also been explored. In 2 patients with an out-of-frame exon 45 deletion treatment with AONs resulted in exon 46 skipping, which should restore the open reading frame. Exon skipping levels were ~15%, which restored the synthesis of functional dystrophin in more than 75% of the cells (van Deutekom et al., 2001). Subsequently, the skipping of different exons has been reported for patient-derived cells with other deletions, point mutations and duplications. For an overview see Aartsma-Rus, *RNA Biology* 2010 (Aartsma-Rus, 2010). Restoration of dystrophin synthesis was detectable at the membrane and the (at least partial) functionality of these BMD-like proteins was suggested by the reformation of the dystrophin-glycoprotein complex, shown by increased membrane expression of DGCassociated proteins. Another interesting finding were the higher exon skipping levels observed in these patient cell lines, than previously seen in control cell lines. This can be explained by nonsense-mediated decay (NMD) of the original out-of-frame transcripts, which are less stable than the newly formed in-frame transcripts (Aartsma-Rus et al., 2003).

In theory, skipping of a single exon would be beneficial for approximately 64% of the known mutations in DMD patients. However, there still is a large population which requires the skipping of 2 or more exons for reading frame restoration (Aartsma-Rus et al., 2009a). The theoretic applicability of exon skipping could be extended to 79% by double exon skipping and around 90% of patients by multiple exon skipping. Feasibility of double exon skipping was first shown in 2 different patient cell lines. One patient had a nonsense mutation in exon 43, for which dystrophin synthesis could be restored by skipping of exon 43 and 44. The second, carrying an exon 46-50 deletion, was successfully treated with a combination of AONs against exon 45 and 51. Dystrophin synthesis was found in 70% of the myotubes, which is almost as high as after single exon skipping (75-80%) (Aartsma-Rus et al., 2004a). Subsequently, successful double exon skipping has been proved by other groups (reviewed in Aartsma-Rus, *RNA Biology* 2010 (Aartsma-Rus, 2010)). The dog model for DMD needs double skipping to bypass the mutation and cells derived from these dogs have been

used to show double exon skipping *in vitro* (see below) (McClorey et al., 2006).

A surprising finding in control myotubes was that combinational treatment with 45AON and 51AON caused the skipping of the entire stretch of exons from 45 through 51. This

**4. Antisense-mediated exon skipping** *in vitro*

**4.1 Single exon skipping** 

patients and in *mdx*-cell cultures.

**4.2 Double and multiple exon skipping** 

would largely increase its therapeutic applicability for a number of different mutations. Indeed the same result could be induced in patient cells with an exon 48-50 deletion (Aartsma-Rus et al., 2004a). Skipping of other large stretches of exons (multiple exon skipping) however turned out to be technically challenging and has had limited success so far (Aartsma-Rus et al., 2006a). The use of several ratios of 45AON and 55AON in both control as patient cell lines resulted in undetectable to very low exon 45-55 skipping frequencies (van Vliet L. et al., 2008).

Exon skipping is in theory useful for the majority of patients. Exceptions are mutations that involve regions in the gene that are essential for the function of the dystrophin protein: all actin-binding N-terminal parts, the cysteine-rich C-terminal part (binding to the DGCcomplex), the promoter region or the first exon. Furthermore it is not applicable to translocations. Fortunately these kind of mutations make up only a small part (~8%) of all known mutations (Aartsma-Rus et al., 2009a). The largest part of mutations is made up by deletions and small mutations. A minor part consists of exon duplications (double or multiple). In the case of single duplications, skipping of one of these exons would in theory generate wild-type dystrophin transcripts. However, this turned out to be challenging. In cells with an exon 45 duplication this was indeed possible, but in other cases the skipping was so efficient that both exons were skipped, leading to an out-of-frame transcript (Aartsma-Rus et al., 2007). Skipping of an additional exon could restore the reading frame again. For example for an exon 18 duplication, successful skipping of exon 17 and both exon 18s resulted in restoration of the reading frame (Forrest et al., 2010). Successful skipping of multiple exon duplications has not yet been achieved (Aartsma-Rus et al., 2007). In total 6% of patients could benefit from single or multiple skipping of exon duplications.
