Scientific background of the exon skip therapy

Duchenne muscular dystrophy is caused by the absence of functional dystrophin protein. This protein fulfills an important linker function and connects the proteins of the skeleton of muscle fibers to the connective tissue that surrounds muscle fibers. Dystrophin acts as a shock absorber and protects muscle fibers against damage during muscle movement (contraction and relaxation of fibers). You can envisage dystrophin as the rope between an anchor and a boat (Figure 1). The anchor can only function when the rope (dystrophin) is connected to both the anchor and the boat.

Figure 1: Dystrophin has an anchor function.

The blueprint of the dystrophin gene is embedded in the DMD (Duchenne muscular dystrophy) gene. The parts containing the actual genetic information the cell needs to generate proteins are called "exons". The DMD gene has 79 exons (Figure 2). These exons fit together like pieces of a jigsaw puzzle and form the genetic code for the dystrophin protein.

Figure 2. The exons of the DMD gene.

Duchenne patients have mistakes (mutations) in their DMD gene. The most common mistake is that one or more exons are missing from the gene (a so called deletion). In the example in Figure 3 exon 48, 49 and 50 are missing (deleted).

Figure 3. A deletion of exon 48-50.

When we zoom in to the part that flanks the mutation, we see that exon 47 does not fit to exon 51 (Figure 4).

Figure 4. Exon 47 does not fit to exon 51.

As exon 47 and exon 51 do not fit, the genetic code is broken. The consequence is that the blueprint becomes unreadable after exon 47 and the translation into dystrophin is stopped prematurely in exon 51. As dystrophin has to connect two different things, this dystrophin is completely non functional (Figure 5).

Figure 5. Due to the mistake in the DMD gene, only the beginning of the dystrophin protein can be made. The connection is completely lost (the part of the rope bound to the boat is not present, so the boat sails off).

Due to the lack of functional dystrophin muscle fibers from Duchenne patients are very sensitive to muscle damage. You are familiar with the consequences...

It is possible that the mutation does not disrupt the genetic code of the DMD gene, as can be seen in an example in Figure 6 and Figure 7.

Figure 6. A deletion of exon 48-51.

Figure 7. Exon 47 fits to exon 52.

Due to the mutation exons 48, 49, 50 and 51 are missing. However, exon 52 fits to exon 47, so the genetic code is maintained and despite the mutation, the blueprint remains readable and the protein synthesis can be continued after the mutation. As the beginning and the end of dystrophin are now present, the connection is not lost, although it is a bit shorter (the part that was described in exon 48-51 is missing). This dystrophin is thus largely functional (Figure 8). The rope is somewhat shorter, but the boat is connected to the anchor and kept in place.

Figure 8. Dystrophin that misses a bit in the middle is partly functional.

Mistakes that do not disrupt the genetic code are found in Becker muscular dystrophy patients. As their dystrophins are partly functional, the muscle fibers are less sensitive to damage than those of Duchenne patients. Generally, the progression of the disease is less severe.
The aim of the exon skip approach is to restore the genetic code for Duchenne patients, so they can make a partly functional dystrophin instead of a non functionl dystrophin. Hopefully this will slow down or even stop disease progression.

Exons can be skipped through so called antisense oligonucleotides (AONs). These are small, synthetic pieces of modified DNA that can cover a specific exon (Figure 9). As a result this exon will be skipped when the genetic code is assembled. In the example of Figure 9 skipping of exon 51 restores the genetic code.

Figure 9. Exon skipping for a patient with a deletion of exon 48-50. Due to the deletion exon 47 is joined to exon 51 when the genetic code is assembled. These exons do not fit and the genetic code is broken (and protein translation is stopped prematurely). Using an AON specific for exon 51 this exon is hidden and skipped during the assembly of the genetic code. Now exon 47 is joined to exon 52, which does fit: the genetic code is restored and protein synthesis can be completed.

Results so far (preclinical)

Which exon has to be skipped to restore the genetic code depends on the mutation. In collaboration with Prosensa we have currently tested over 150 AONs and identified AONs to induce skipping of 39 DMD exons. In theory this can restore the genetic code for ~85% of all Duchenne patients. As some mutations are more common than others, the skipping of certain exons is applicable to relatively large groups of patients. The best example would be exon 51 skipping, which is applicable to 16% of all patients. Other exons applicable to large groups are exon 45 (11%), exon 53 (9.5%) and exon 44 (8%).
We have tested our AONs in cultured muscle cells derived from Duchenne patients. For each patients we observed after AON treatment that the targeted exon was skipped and partly functional dystrophin was produced. In addition, in the mdx mouse model (which has a mutation in the mouse dystrophin gene that disrupts the genetic code) AON treatment also results in exon skipping and dystrophin restoration. This was accompanied by improved muscle function compared to non treated mdx mice.

Results of the first clinical study

Due to the encouraging preclinical results, Prosensa and the LUMC (Center for Human and Clinical Genetics and Department of Neurology) have set up a clincial trial to test this approach in Duchenne patients. The study involved four patients who received a single local injection with an AON targeting exon 51 (PRO051) in their shin muscle (tibialis anterior). A muscle biopsy was taken a month after injection from the treated area. For each patients exon 51 was skipped and dystrophin production restored in the majority of muscle fibers in the biopsy. In addtion, the treatment was well tolerated by the patients (there were no serious adverse events). These results where beyond what we had dared hope for and are very encouraging for subsequent studies.

And now?

The first clinical trial was required to show that the exon skip approach worked in patients and was safe and tolerated. However, local treatment of AONs to all individual muscles is not feasible. Therefore, current research by the LUMC and Prosensa focuses on ways to optimize systemic treatments in the mdx mouse model. In addition, Prosensa is preparing for clinical trials where PRO051 will be tested systemically, and developing AONs to induce skipping of other exons (see www.prosensa.nl for more information).

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Scientific background of the exon skip therapy

Figure 2. The exons of the DMD gene.

Duchenne patients have mistakes (mutations) in their DMD gene. The most common mistake is that one or more exons are missing from the gene (a so called deletion). In the example in Figure 3 exon 48, 49 and 50 are missing (deleted). Figure 3. A deletion of exon 48-50.

When we zoom in to the part that flanks the mutation, we see that exon 47 does not fit to exon 51 (Figure 4).

Figure 4. Exon 47 does not fit to exon 51.

Figure 5. Due to the mistake in the DMD gene, only the beginning of the dystrophin protein can be made. The connection is completely lost (the part of the rope bound to the boat is not present, so the boat sails off).

Due to the lack of functional dystrophin muscle fibers from Duchenne patients are very sensitive to muscle damage. You are familiar with the consequences...

It is possible that the mutation does not disrupt the genetic code of the DMD gene, as can be seen in an example in Figure 6 and Figure 7.

Figure 6. A deletion of exon 48-51.

Figure 7. Exon 47 fits to exon 52.

Figure 8. Dystrophin that misses a bit in the middle is partly functional.

Results so far (preclinical)

Results of the first clinical study

And now?

Duchenne patients have mistakes (mutations) in their DMD gene. The most common mistake is that one or more exons are missing from the gene (a so called deletion). In the example in Figure 3 exon 48, 49 and 50 are missing (deleted).

Figure 3. A deletion of exon 48-50.