Background of the Disease

The progressive deterioration of muscle fibers in Duchenne Muscular Dystrophy (DMD, OMIM#310200) is caused by the deficiency of dystrophin. This is a protein that is important for the strength and flexibility of the muscle fiber membranes (detailed information). The genetic code (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 (mutations). In particular, deletions of one or more exons (~65%), but also exon duplications (~7%), and small point mutations (~25%) have been identified (see the DMD/BMD mutation database at www.dmd.nl or this paper ). Many mutations in the DMD gene disrupt the open reading frame and thus cause the premature abortion of the synthesis of the dystrophin, leading to the severe DMD phenotype (reading frame further explained). 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. 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.

                          

 

Background of the Technology

Over the last 10 to 15 years much effort has been put into the development of a safe and efficient gene therapy for DMD. However, the DMD gene and its product initially seemed too large and too complex to allow a straightforward approach. Over the last few years an innovative tool has emerged with which an escape route can be utilized that nature had already hinted at. Some DMD patients have rare, dystrophin-positive fibres (“revertant fibers”), originating from reading frame-restoring exon skipping. Several laboratories have recently shown that we can actually enhance or induce this therapeutic exon skipping using small synthetic antisense oligoribonucleotides (AONs).

Through inducing the skipping of exons during the splicing, AONs can restore the reading-frame of the dystrophin transcript (mRNA), and thus convert DMD into BMD-like fibers. AONs vary in length between 16 and 22 nucleotides and are chemically modified to be resistant to intracellular nucleases. They are suggested to bind to specific sequences in the pre-mRNA, and thus disturb exon inclusion signals like splice sites, intronic branch point sequences, or exonic splicing enhancer elements. This leads to the removal of the targeted exon.

 

                   

 

The generally applied procedure for the analysis of therapeutic exon skipping in cultured muscle cells is as follows. Muscle cells derived from DMD patients are proliferated in culture, and then allowed to differentiate into multinucleated myotubes through serum-deprivation.  These myotube cultures are transfected with a sequence-specific, exon-internal AON with which the skipping of a specific exon can be induced. For transfection we use the cationic polymer polyethylenimine (PEI) which is very efficient in delivering AONs into myotubes (up to 95% transfection efficiencies) (Figure 1). After 24 to 72 hours RNA is isolated from the treated cultures and analysed by RT-PCR (see Figure 2). Correct exon skipping in the smaller transcript fragment is confirmed by sequencing. Immunohistochemical analyses with different dystrophin antibodies is performed to novel dystrophin expression at the membrane (Figure 3). In addition, total protein samples are isolated to detect dystrophin by Western Blot analysis  (Figure 4).

 

                Figure 1. PEI Transfection of myotube cultures using a fluorescence-labeled AON (back to text)

               

                 

Figure 2. RT-PCR Analysis to detect specific exon (46) skipping in RNA samples (back to text)

 

 

Figure 3. Immunohistochemical Analysis to detect dystrophin at the membranes of myotubes (back to text)

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Western Blot Analysis to detect dystrophin in protein samples (back to text)