Development of an anitsense-mediated exon skipping therapy for Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a severe, lethal neuromuscular disorder caused by reading frame disrupting mutations (mostly deletions) in the dystrophin gene. This results in the complete absence of dystrophin and leads to the continuous loss of muscle fibers and fibrosis. As a consequence, DMD patients are wheelchair dependent before the age of 12 and often die in the third decade of the life (or earlier) due to respiratory- or heart failure. Deletions in the dystrophin gene that keep the reading frame intact allow the generation of internally deleted, partly functional dystrophins and are associated with the milder Becker muscular dystrophy (BMD). Becker patients often remain ambulant until later in life and have near normal life expectancies. Normal dystrophin consists of an N-terminal actin-binding domain, a central rod domain (containing 24 spectrin-like repeat units and 4 hinge regions) a cysteine-rich and a C-terminal domain. Dystrophin is thought to fulfill a bridge function between the cytoskeleton and the extracellular matrix, since the actin binding domain binds to cytoskeletal actin, while the C-terminal domain is involved with the transmembranal dystrophin glycoprotein complex (DGC) that is connected to the extracellular matrix via laminin 2. In DMD patients this bridge function is completely lost, since the C-terminal bridgehead is lacking due to a truncating mutation. In BMD patients on the other hand, an internal deletion results in a shorter, but still semi-functional bridge that contains both the N-terminal and C-terminal bridgeheads. As yet there is no clinically applicable therapy for DMD patients, despite extensive research for a variety of different approaches. Currently, one of the most promising strategies is the antisense-mediated reading frame restoration. The aim of this approach is to induce specific exon skipping to convert an out of frame DMD transcript into its nearest in frame BMD-like counterpart. This would allow the generation of an internally deleted but partly functional dystrophin and should convert DMD into a milder BMD phenotype. The skipping of a specific exon can be induced by antisense oligoribonucleotides (AONs), which are small synthetic RNAs. Upon binding of the AONs to the pre-mRNA the splicing machinery does not recognize the exon as such anymore, and as a result the targeted exon is spliced out with its flanking introns (i.e. the exon is "skipped"). The broad mutation spectrum found for DMD would require the skipping of a series of exons to restore the reading frame for several patients. Fortunately, designing efficient DMD specific AONs has proven relatively easy, and we can currently induce the specific skipping of 20 different exons in human control myotube cultures after PEI-mediated AON delivery (Chapter 2). This would restore the reading frame for over 40% of all DMD patients. The broad therapeutic applicability of this technique was confirmed in myotube cultures derived from 8 different patients (Chapter 3). For each patient skipping of the specific exons could be induced on RNA level and dystrophin synthesis was restored in over 75% of treated myotubes. Time course experiments revealed that dystrophin was detectable as early as 16 hours post transfection and increasing levels were found for up to 7 days. In addition, expression of DGC proteins was restored in treated myotube cultures, further confirming the functionality of the BMD-like dystrophins. Since a significant part of DMD patients carries a mutation that requires the skipping of two exons, we also tested the feasibility of double-exon skipping in two patients (Chapter 4). After treatment with a mix of the respective AONs, double exon skipping was detected on RNA level and dystrophin synthesis was restored for over 70% of treated myotube cultures for both patients. Furthermore, when we treated control myotubes with AONs targeting exons 45 and 51 we observed multi-exon skipping of exon 45 through 51. Multi-exon skipping not only increases the applicability of this technique, it also reduces the mutation specificity, since it allows for the generation of a BMD-like deletion that covers the majority of DMD mutations. The skipping of exon 45 through 51 would already be applicable to 15% of all patients and its feasibility was confirmed in myotubes derived from a patient carrying an exon 48-50 deletion. Subsequent experiments aiming at the skipping of a larger number of exons seem to indicate that the number of exons that can be skipped is limited due to hitherto unknown processes. Thus far we have used AONs containing 2'-O-methyl RNA with a full-length phosphorothioate backbone (2OMePS), which are cytotoxic at high concentrations. For future clinical applications the optimal AON induces high levels of specific exon skipping at low levels of cytotoxicity. We thus compared the efficacy and efficiency of our most efficient exon 46 2OMePS AON to those of a morpholino, a locked nucleic acid (LNA) and a peptide nucleic acid (PNA) AON (Chapter 5). Only the LNA induced higher levels of exon skipping than 2OMePS in patient and control myotube cultures. However, when we compared the sequence specificity of these analogues we observed that LNAs appear to be much less sequence specific than 2OMePS AON. Therefore, we conclude that 2OMePS currently seem the favorable compounds to establish clinical trials. To study exon skipping in vivo we injected PEI-coupled 2OMePS AONs specific for murine exon 46 into the gastrocnemius muscle of normal mice (Chapter 6). Relatively low levels of exon 46 skipping could be detected on RNA level and persisted for over four weeks post injection. Furthermore, we have previously engineered a mouse model that contains the entire human DMD gene (2.6 Mb) integrated into the murine genome (hDMD mouse). These transgenic mice uniquely allow for the preclinical testing of human-specific AONs in vivo. We have injected AONs targeting human exons 44, 46 and 49 into the musculus. gastrocnemicus of hDMD mice, and showed that the skipping of the human exons (but not the murine exons) was indeed specifically induced. Based on pre-clinical data obtained by our group and others, we are currently setting up a clinical trial aiming at local dystrophin restoration following intramuscular injections of exon 46 and 51 specific AONs. For future application, however, we aim at systemic delivery of AONs. Therefore, we are currently investigating delivery methods that will allow systemic delivery of AONs.