The progressive deterioration of muscle fibers in Duchenne Muscular Dystrophy (DMD, OMIM#310200) is caused by the deficiency of the dystrophin protein. This is a protein that is important for the strength and flexibility of the muscle fiber membranes. The genetic code, also known as a reading frame, for dystrophin is encoded by the so-called DMD gene. This is the largest human gene with 79 exons (coding units) dispersed over an enormous stretch of DNA of no less than 2.4 million base pairs.
The exons are put together for code assembly during a process called splicing. Due to its length the DMD gene is relatively vulnerable for rearrangements, the so called mutations. In particular, deletions of one or more exons (~65%), but also exon duplications (~7%), and small point mutations (~25%) have been identified (see DMD/BMD mutation database).
Many mutations in the DMD gene disrupt the open reading frame and thus cause the premature abortion of the synthesis of dystrophin, leading to the severe DMD phenotype. However, numerous other mutations are “in-frame”, and conserve the reading frame. Despite for instance a large internal deletion, a truncated but mostly functional dystrophin is produced (Figure 1). In these patients, with Becker muscular dystrophy (BMD), the extent and progression of the muscle weakness is less severe. BMD patients thus show intermediate to milder phenotypes with much longer to normal life expectancies.
Different steps take place before a gene is translated into a protein. First a temporary gene copy is produced (RNA transcript). For most genes the genetic code is dispersed. For these genes, the pieces containing the genetic code (exons) have to be joined together and the pieces with non containing genetic information (introns) have to be removed. This process is called splicing and results in a messenger RNA that contains only the genetic code and is translated into protein by the cell.
Exon skipping aims to restore the genetic code from Duchenne patients during the splicing process, so a partially functional, Becker-like dystrophin protein can be made, rather than a non-functional Duchenne protein. This restoration is achieved by the AONs (antisense oligonucleotides). AONs are small pieces of modified RNA that recognize a target exon, bind to it and hide it from the splicing machinery. This results in the skipping of that specific exon and the restoration of the genetic code, so AON treatment is resulting in the production of Becker-like dystrophins (proteins). This approach was first developed in patient-derived cultured cells (in vitro studies) and the mdx mouse model (in vivo studies). This resulted in exon skipping and dystrophin restoration. The approach then moved into the clinical trial phase to be tested in patients. Clinical trials are divided in two phases: the early phase trials are primarily done to show that the approach is safe, while in later stage trials the goal is to show that the treatment is effective as well as safe. All information obtained in studies in cells, animals and especially in patients is used to apply for market authorization, which is required before the drug can be brought on the market.