**7. Improvement of AON delivery and efficiency**

The efficacy of AONs depends partly on the amount of AON that reaches its target, i.e. the muscle fibre nuclei. Several strategies to improve muscle-specific uptake are under investigation, like muscle-homing peptides and cell-penetrating peptides (see above). Due to AON clearance and turnover, the effect of AONs is only temporarily, thus repeated, life-long, injections are required, should this approach prove to be efficacious. The first clinical trials showed that the average serum half-life was 29 days for 2OMePS AONs and around 1.5 hours for PMOs. A way to allow a more prolonged effect is the use of viral vectors stably expressing modified small nuclear ribonucleoprotein (snRNP) genes, in which the normal antisense sequence is replaced by an antisense sequence of choice. snRNPs are small protein-RNA hybrids that are amongst others involved in pre-mRNA splicing and histone processing. The U1 and U7 snRNPs have been used most in splicing modulation experiments (Brun et al., 2003). Exon 51 targeting U1 snRNPs induced effective skipping of exon 51 and rescue of dystrophin synthesis in a patient-derived cell line (De Angelis et al., 2002). Adeno-associated viruses (AAVs) are very efficient at transferring genes into skeletal muscles. Injection of AAV vectors expressing U7 or U1 snRNPs targeting mouse exon 23 resulted in sustained production of functional dystrophin in the *mdx* mouse after intramuscular injection and body-wide dystrophin expression and reduced muscle wasting after systemic treatment (Denti et al., 2008; Goyenvalle et al., 2004). However serious problems with the use of AAV vectors are the possibility of an immune response against the viral capsid and the difficulty to produce them on a large scale under good manufacturing practice (GMP), necessary for implementation in

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 73

DMD gene. *J.Biol.Chem.*, Vol.283,No.9, (February 2008), pp. 5899-5907, ISSN Aartsma-Rus, A. (July 2010). Antisense-mediated modulation of splicing: therapeutic

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Aartsma-Rus, A., Fokkema, I., Verschuuren, J., Ginjaar, I., van, D. J., van Ommen, G. J., &

Aartsma-Rus, A., Janson, A. A., Kaman, W. E., Bremmer-Bout, M., Den Dunnen, J. T., Baas,

Aartsma-Rus, A., Janson, A. A., Kaman, W. E., Bremmer-Bout, M., van Ommen, G. J., Den

Aartsma-Rus, A., Janson, A. A., van Ommen, G. J., & van Deutekom, J. C. (2007). Antisense-

Aartsma-Rus, A., Kaman, W. E., Bremmer-Bout, M., Janson, A. A., Den Dunnen, J. T., van

cells. *Gene Ther.*, Vol.11,No.18, (September 2004b), pp. 1391-1398, ISSN Aartsma-Rus, A., Kaman, W. E., Weij, R., Den Dunnen, J. T., van Ommen, G. J., & van

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implications for Duchenne muscular dystrophy. *RNA.Biol.*, Vol.7,No.4, (July 2010),

Dunnen, J. T., & van Deutekom, J. C. (December 2005). Functional analysis of 114 exon-internal AONs for targeted DMD exon skipping: indication for steric hindrance of SR protein binding sites. *Oligonucleotides.*, Vol.15,No.4, (December

Den Dunnen, J. T. (March 2009a). Theoretic applicability of antisense-mediated exon skipping for Duchenne muscular dystrophy mutations. *Hum.Mutat.*,

F., van Ommen, G. J., & van Deutekom, J. C. (April 2003). Therapeutic antisenseinduced exon skipping in cultured muscle cells from six different DMD patients.

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induced exon skipping for duplications in Duchenne muscular dystrophy.

Ommen, G. J., & van Deutekom, J. C. (September 2004b). Comparative analysis of antisense oligonucleotide analogs for targeted DMD exon 46 skipping in muscle

Deutekom, J. C. (September 2006a). Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. *Mol.Ther.*, Vol.14,No.3, (September 2006a), pp. 401-407, ISSN Aartsma-Rus, A., van Deutekom, J. C., Fokkema, I. F., van Ommen, G. J., & Den Dunnen, J.

T. (August 2006b). Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. *Muscle Nerve.*, Vol.34,No.2, (August 2006b), pp. 135-144, ISSN Aartsma-Rus, A. and van Ommen, G. J. (October 2009). Less is more: therapeutic exon

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mechanisms. *Mol.Ther.*, Vol.17,No.3, (March 2009b), pp. 548-553, ISSN Alter, J., Lou, F., Rabinowitz, A., Yin, H., Rosenfeld, J., Wilton, S. D., Partridge, T. A., & Lu,

the clinic. Another problem is the translation from mice to larger animals or humans. In mice it is feasible to treat a whole muscle, but transfection of whole muscles body-wide is more challenging in larger animals and humans.

#### **8. Conclusion**

In summary, Duchenne muscular dystrophy is caused by genetic defects in the gene encoding the dystrophin protein. These mutations cause a premature stop codon or disrupt the reading frame, leading to a non-functional protein. In most cases this can be overcome by specific skipping of the mutated exon with AONs, to produce a slightly shorter, but largely functional dystrophin protein, as found in the related, but much milder Becker muscular dystrophy. Over the past years major steps have been made in development of this therapy. Proof-of-principle has first been shown *in vitro* in cultured muscle cell lines and *in vivo* in several animal models (e.g. *mdx* mice and GRMD dogs). Recently the first clinical trials with AONs of 2 different chemistries, targeting exon 51, applicable to the largest group of patients, have been completed with positive results. Larger trials are ongoing or planned for the near future. Although the results obtained in the past few years are very encouraging, precaution is needed and several problems still exist. First of all, this is not a cure, but a potential treatment that will hopefully lead to an improvement of the phenotype. Secondly, the approach is mutation-specific, i.e. requiring different AONs for different mutations. Luckily most mutations cluster in 2 hotspots (see above). However, development and application in the clinic of the therapy for rare mutations will be difficult, since at the moment each AON is considered as a new drug, therefore has to go through all (pre)clinical steps before it can be registered. For these rare mutations simply not enough patients are available for these studies. At the moment efforts to discuss this with the regulatory authorities are coordinated by the TREAT-NMD Network of Excellence. For example, it may be possible to reduce the toxicity trials for an AON with similar backbone chemistry, if 1 or 2 of this kind have been proven to be safe (Muntoni & Wood, 2011). Thirdly, the approach will not be useful for mutations affecting the essential parts (actin-or dystroglycan-binding domains) of the protein. Fortunately these make up only a small percentage of all known mutations. Furthermore, restoration of the reading frame is more challenging when double and especially multiple exon skipping is required. Finally, the preclinical studies and first clinical trials have shown that muscle quality is very important for the therapeutic success, since dystrophin transcripts are only produced in muscle cells and not in the fibrotic and adipose tissue that replaces the muscle cells when the disease progresses. Therefore early start of treatment will probably be required.

In conclusion, AONs are currently a promising therapeutic approach for DMD and major steps towards clinical implementation have been made over the past years, but further improvements are necessary for increasing therapeutic effectiveness and more research for broader clinical application of the technique.

#### **9. References**

't Hoen, P. A., de Meijer, E. J., Boer, J. M., Vossen, R. H., Turk, R., Maatman, R. G., Davies, K. E., van Ommen, G. J., van Deutekom, J. C., & Den Dunnen, J. T. (February 2008).

the clinic. Another problem is the translation from mice to larger animals or humans. In mice it is feasible to treat a whole muscle, but transfection of whole muscles body-wide is more

In summary, Duchenne muscular dystrophy is caused by genetic defects in the gene encoding the dystrophin protein. These mutations cause a premature stop codon or disrupt the reading frame, leading to a non-functional protein. In most cases this can be overcome by specific skipping of the mutated exon with AONs, to produce a slightly shorter, but largely functional dystrophin protein, as found in the related, but much milder Becker muscular dystrophy. Over the past years major steps have been made in development of this therapy. Proof-of-principle has first been shown *in vitro* in cultured muscle cell lines and *in vivo* in several animal models (e.g. *mdx* mice and GRMD dogs). Recently the first clinical trials with AONs of 2 different chemistries, targeting exon 51, applicable to the largest group of patients, have been completed with positive results. Larger trials are ongoing or planned for the near future. Although the results obtained in the past few years are very encouraging, precaution is needed and several problems still exist. First of all, this is not a cure, but a potential treatment that will hopefully lead to an improvement of the phenotype. Secondly, the approach is mutation-specific, i.e. requiring different AONs for different mutations. Luckily most mutations cluster in 2 hotspots (see above). However, development and application in the clinic of the therapy for rare mutations will be difficult, since at the moment each AON is considered as a new drug, therefore has to go through all (pre)clinical steps before it can be registered. For these rare mutations simply not enough patients are available for these studies. At the moment efforts to discuss this with the regulatory authorities are coordinated by the TREAT-NMD Network of Excellence. For example, it may be possible to reduce the toxicity trials for an AON with similar backbone chemistry, if 1 or 2 of this kind have been proven to be safe (Muntoni & Wood, 2011). Thirdly, the approach will not be useful for mutations affecting the essential parts (actin-or dystroglycan-binding domains) of the protein. Fortunately these make up only a small percentage of all known mutations. Furthermore, restoration of the reading frame is more challenging when double and especially multiple exon skipping is required. Finally, the preclinical studies and first clinical trials have shown that muscle quality is very important for the therapeutic success, since dystrophin transcripts are only produced in muscle cells and not in the fibrotic and adipose tissue that replaces the muscle cells when the disease progresses. Therefore early

In conclusion, AONs are currently a promising therapeutic approach for DMD and major steps towards clinical implementation have been made over the past years, but further improvements are necessary for increasing therapeutic effectiveness and more research for

't Hoen, P. A., de Meijer, E. J., Boer, J. M., Vossen, R. H., Turk, R., Maatman, R. G., Davies, K.

E., van Ommen, G. J., van Deutekom, J. C., & Den Dunnen, J. T. (February 2008).

challenging in larger animals and humans.

start of treatment will probably be required.

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**4** 

*Canada*

**Psychosocial Support** 

Jean K. Mah1,\* and Doug Biggar2

**Needs of Families of Boys with Duchenne Muscular Dystrophy** 

*2Bloorview Kids Rehab, University of Toronto, Ontario,* 

*1Alberta Children's Hospital, University of Calgary, Calgary, Alberta,* 

Duchenne muscular dystrophy (DMD, OMIM #310200) is the most common form of muscular dystrophy in childhood, with an incidence of approximately 1 per 3,500 live-born males [Emery, 1991]. It is caused by mutations of the *DMD* gene located on Xp21 which codes for dystrophin, a 427-kDa protein that is expressed at the sarcolemma of skeletal muscle. The *dystrophin* gene contains 79 exons, which includes an actin-binding domain at the N-terminus, 24 spectrin-like repeat units, a cysteine-rich dystroglycan binding site, and a C-terminal domain [Hoffman et al, 1987; Koenig et al, 1988]. The large size of the *dystrophin* gene results in a complex mutational spectrum (>4,700 different mutations) as well as a high spontaneous mutation rate [Aartsma-Rus et al, 2006]. Large deletions account for approximately 65% of DMD mutations while duplications occur in up to 10% of males with DMD. The remaining 25% include small deletions, insertions, point mutations, or splicing mutations. About two-thirds of DMD cases are inherited from mothers carrying the mutations, with the remaining one-third occurring as spontaneous mutations [Laing, 1993]. According to Monaco et al, DMD-causing mutations are typically associated with an out-offrame mutation leading to a loss of functional gene product, whereas in-frame mutations that allow synthesis of an internally truncated but functional protein result in a milder

Dystrophin is an integral component of the dystrophin glycoprotein complex. It stabilizes the muscle membrane by bridging the basal lamina of the extracelluar matrix to the inner cytoskeleton of the contractile elements [Rybakova et al, 2000]. It also serve as a transmembrane signalling complex which is essential for cell survival [Chen et al, 2000]. Loss of dystrophin results in excessive membrane fragility, unregulated influx of calcium ions into the sarcoplasm, mitochondrial dysfunction, and increased oxidative stress, leading to progressive muscle degeneration, fibrosis, and fatty replacement [Wallace & McNally, 2009]. Early presenting features in DMD include developmental delay, proximal muscle weakness as evident by Gowers' sign and waddling gait, as well as varying degree of

Becker muscular dystrophy (BMD) phenotype [Monaco et al, 1988].

**1. Introduction** 

 \*

Corresponding Author

skipping and functional restoration in dystrophin-deficient mdx mice. *Hum.Mol.Genet.*, Vol.18,No.22, (November 2009), pp. 4405-4414, ISSN

