**5. Gene therapy and gene editing for MDs**

In previous decades, significant advancements in direct gene replacement approaches in genetic muscle diseases have been achieved. Two strategies are currently being tested in the dystrophic animal model and have already entered - or are ready to enter - clinical experi‐ mentation. These are exon skipping and the expression of dystrophin variants of reduced size (Figure 2).

For the exon-skipping experiments, adeno associated viral vectors (AAV) were engineered to produce small nuclear U7 RNA targeting exons [28, 29]. These excluded the mutation of dystrophin from an in-frame transcript that is translated in a 'quasi' normal dystrophin protein. High-pressure intravenous delivery was adopted to guarantee an efficient systemic delivery. Phase I and II clinical protocol in patients was designed by scaling up AAV produc‐ tion for total body delivery and transient immune suppression to enable reinfusions.

As an alternative strategy, dystrophin variants of reduced size were considered, since the large size of the transcripts (14 kb) is an impediment to generate viral vectors. Indeed, a mild phenotype was observed in Becker muscular dystrophy patients, characterized by a huge deletion of dystrophin gene. This resulted in the expression of truncated, yet partially func‐ tional, dystrophin. This observation led to the idea of using truncated dystrophins for therapeutic use in mdx mice via rAAV vector-mediated gene transfer (for a detailed review see [30]). Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer repre‐ sents a promising approach for genetic diseases of muscles. Despite the limited DNA pack‐ aging capacity (~4.8 kb), its transduction efficiency is very impressive. Recently, Chamberlain group showed that functional dystrophin transgenes could be reconstituted *in vivo* by homologous recombination (HR), following intravascular co-delivery of two independent rAAV6 vectors [31]. Systemic delivery of dystrophin variants of reduced size has also been effective in pigs (Pichavant et al., 2010). Unfortunately, a phase 1/2 clinical trial using intra‐ muscular injections of AAV2 into DMD patients did not result in the restoration of dystrophin expression. This is likely due to T cell immunity to dystrophin proteins. An alternative strategy is the delivery of utrophin, a dystrophin related protein normally present at neuromuscular junctions, which should not elicit an immune response. Utrophin has 3,433 amino acids, with a predicted molecular mass of 395 kDa. It is slightly smaller than dystrophin (427 kDa) and ubiquitously expressed. In addition, while dystrophin is expressed throughout the sarcolem‐ ma of skeletal muscle fibres, utrophin is restricted at the neuromuscular and myotendinous junction.

A significant inverse correlation between utrophin expression and disease severity in DMD has been observed [32]. Davies group developed a range of strategies to up-regulate utrophin for therapeutic approaches in muscular dystrophies [33]. A number of drugs were tested in a stable H2K mdx myoblast cell line. Here, a luciferase reporter gene was under control of the mouse utrophin promoter to identify an effect on utrophin expression [34]. This long screening study allowed the identification of SMT C1100, which also showed therapeutic potential in the mdx mouse [35]. Since the drug also demonstrated a synergistic effect when administered with prednisolone [35], the gold standard in treatment of DMD patients in the clinic, SMT C1100 was tested in phase I trials by BioMarin Pharmaceuticals (as BMN-195; Novato, CA, USA). Unfortunately, the plasma levels of the drug were not high enough for the trials to continue. Although there were no safety issues, new formulations are necessary.

wasting in muscle mass and force. Insulin-like Growth Factor-1 (IGF-1), MAGIC-F1 and myostatin regulate the key steps during muscle regeneration (Figure 3). In animal models for Duchenne muscular dystrophy [22, 25, 27], these molecules have demonstrated a therapeutic value, without redressing the primary cause of the lesion and, in principle, could be adopted in patients suffering from muscular dystrophies. The delivery strategies of these molecules and potential side effects require more investigation. So far, their translational potential has

In previous decades, significant advancements in direct gene replacement approaches in genetic muscle diseases have been achieved. Two strategies are currently being tested in the dystrophic animal model and have already entered - or are ready to enter - clinical experi‐ mentation. These are exon skipping and the expression of dystrophin variants of reduced size

For the exon-skipping experiments, adeno associated viral vectors (AAV) were engineered to produce small nuclear U7 RNA targeting exons [28, 29]. These excluded the mutation of dystrophin from an in-frame transcript that is translated in a 'quasi' normal dystrophin protein. High-pressure intravenous delivery was adopted to guarantee an efficient systemic delivery. Phase I and II clinical protocol in patients was designed by scaling up AAV produc‐

As an alternative strategy, dystrophin variants of reduced size were considered, since the large size of the transcripts (14 kb) is an impediment to generate viral vectors. Indeed, a mild phenotype was observed in Becker muscular dystrophy patients, characterized by a huge deletion of dystrophin gene. This resulted in the expression of truncated, yet partially func‐ tional, dystrophin. This observation led to the idea of using truncated dystrophins for therapeutic use in mdx mice via rAAV vector-mediated gene transfer (for a detailed review see [30]). Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer repre‐ sents a promising approach for genetic diseases of muscles. Despite the limited DNA pack‐ aging capacity (~4.8 kb), its transduction efficiency is very impressive. Recently, Chamberlain group showed that functional dystrophin transgenes could be reconstituted *in vivo* by homologous recombination (HR), following intravascular co-delivery of two independent rAAV6 vectors [31]. Systemic delivery of dystrophin variants of reduced size has also been effective in pigs (Pichavant et al., 2010). Unfortunately, a phase 1/2 clinical trial using intra‐ muscular injections of AAV2 into DMD patients did not result in the restoration of dystrophin expression. This is likely due to T cell immunity to dystrophin proteins. An alternative strategy is the delivery of utrophin, a dystrophin related protein normally present at neuromuscular junctions, which should not elicit an immune response. Utrophin has 3,433 amino acids, with a predicted molecular mass of 395 kDa. It is slightly smaller than dystrophin (427 kDa) and ubiquitously expressed. In addition, while dystrophin is expressed throughout the sarcolem‐ ma of skeletal muscle fibres, utrophin is restricted at the neuromuscular and myotendinous

tion for total body delivery and transient immune suppression to enable reinfusions.

been hindered in clinical trials.

(Figure 2).

400 Muscle Cell and Tissue

junction.

**5. Gene therapy and gene editing for MDs**

In conclusion, the systemic delivery of AAV, plasmids and molecules to counteract muscle muscular dystrophy still face significant technical hurdles and alternative strategies are necessary.
