Exon skip technique

The aim of exon skipping is to restore the genetic code, so that the correct protein subunits are used (instead of the aberrant ones), to allow the synthesis of a partially functional proteins (a model car that has some parts missing is more useful than one with aberrant airplane parts). The reasoning for this approach is explained in the ‘backgrond DMD’ page in detail. Exon skipping can be used to restore the genetic code for different mutations types.

The following figures contain three parts: at the top the situation at DNA level (so the level of the gene) is depicted, in the middle the RNA copy is shown (occasionally one of the exons is hidden by an AON). At the bottom the resulting mRNA (so only the genetic code (the exons) is shown.

Deletions

For most deletions of one or more exons, the genetic code can be restored by the skipping of one (Figure 1) or two (Figure 2) exons.

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Figure 1. In this example exon 48-50 as missing (due to a deletion). As exon 47 and 51 do not fit, the genetic code is broken. Since exon 47 and 52 do fit, the genetic code can be restored by skipping of exon 51.

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Figure 2. In this example exon 46-50 are deleted. Exon 45 and exon 51 do not fit, so the genetic code is broken. However, skipping of a single additional exon will not restore the genetic code (exon 45 skipping does not improve things, as exon 45 and 52 do not fit either, and exon 51 skipping also does not work, as exon 45 and 52 do not fit). Only when both exon 45 and exon 52 are skipped, the genetic code is restored (exon 44 fits to exon 52).

Small mutations

Small mutations can result in a premature stop signal (stop mutations), or they can disrupt the genetic code (a small deletion or duplication within and exon). Using exon skipping; these mutations can be bypassed (Figure 3 and 4).

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Figure 3. Due to a small mutations, a premature stop signal has arisen in exon 49. Because exon 48 and exon 50 fit, skipping exon 49 will bypass the stop signal without disrupting the genetic code.

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Figure 4. Due to a small mutation, a stop signal occurs in exon 11. Exon 10 and 12 do not fit, so skipping exon 11 will bypass the stop signal, but also disrupt the genetic code. Exon 10 does fit to exon 13, so skipping both exon 11 and exon 12 will bypass the stop signal, while maintaining the genetic code.

Duplications

Duplications disrupt the genetic code because one or more exons are duplicated. This complicates exon skipping, as the AONs can not distinguish the duplicated and the original exon (they are completely similar). For duplications involving a single exon, exon skipping can be used to restore the genetic code (figures 5, 6 and 7). However, for larger duplications, exon skipping is currently too complicated (figures 8, 9 and 10).

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Figure 5. A duplication of exon 45. As the additional exon 45 (white/red) does not fit to the original exon 45 (blue) the genetic code is broken. However, skipping either the duplicated or the original exon will restore the genetic code completely (this is the normal genetic code: no exons are missing). This is the most ideal result that can be obtained through exon skipping. However, sometimes exon skipping is too efficient (figure 6). The efficiency of exon skipping is difficult to predict and only becomes apparent when AONs are tested in cultured muscle cells from individual patients with a single exon duplication.

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Figure 6. When exon 45 skipping is too efficient for a patient with an exon 45 duplication, both the original and the duplicated exon will be skipped. As exon 44 does not fit to exon 46 this will disrupt the genetic code.

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Figure 7. If skipping a single exon is not feasible (figure 6) because exon skipping is too efficient, the genetic code can still be restored by skipping an additional (third) exon (exon 44 in this example). Since exons 43 and 46 fit, this will restore the genetic code. (In this example it is also feasible to skip exon 46 in addition to both exons 45, since exon 44 and 47 also fit).

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Figure 8. Exon skipping for larger duplications is complicated. In this example, exons 44-48 are duplicated. This disrupts the genetic code, as exon 44 and exon 48 do not fit. The genetic code can be restored through skipping of the duplicated exon 44. However, an AON targeting exon 44 will also recognize the original exon 44 (figure 9 and 10), which will disrupt the genetic code (regardless of whether the duplicated exon is skipped).

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Figure 9. Skipping the original exon 44 disrupts the genetic code, as exon 43 and 45 do not fit.

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Figure 10. When both the original and the duplicated exon are skipped the genetic code is broken as well. There are three possibilities (skipping both exons 44, skipping the original exon 44 and skipping the duplicated exon 44) and only one of them (skipping the duplicated exon 44) is beneficial. Therefore, the efficiency of AON treatment will be very low.