**2.** *DMD* **gene and dystrophin protein**

#### **2.1 Genetic defect in DMD**

DMD is caused by a genetic defect in the *DMD* gene. In approximately 33% of cases this is a de novo (new) mutation. The *DMD* gene is located on the short arm of the X-chromosome (at Xp21). It is the largest gene in the human genome consisting of 2 220 223 base pairs. The coding sequence spans around 0.5% (11 058 bases) of the gene, dispersed over 79 exons. Mutations in the gene causing a disruption of the open reading frame or introducing a premature stop codon lead to a complete absence of a functional dystrophin protein. Dystrophin consists of 3 685 amino acids and has a molecular weight of 427 kDa (Muntoni et al., 2003). The protein is located inside the muscle fibres and forms a bridge between the actin cytoskeleton and the extracellular matrix (ECM). Thereby it provides mechanical stability to the muscle fibres during each contraction. The protein consists of 4 domains: first an N-terminus, containing 2 actin-binding domains (ABD), both consisting of a CH1-and a CH2-domain, which are bound to contractile structures (F-actin) inside the muscle cells. This is followed by a central domain, so called central rod domain, consisting of 24 spectrin-like triple helical coiled repeat units, interrupted by 4 proline-rich hinge regions. A third actinbinding domain is present between repeat 11 and 17 (Amann et al., 1998), while repeat 16-17 contain a binding site for neuronal nitric oxide synthase (nNOS) (Lai et al., 2009). Subsequently the protein contains a cysteine-rich part and finally a C-terminal domain. The cysteine-rich domain binds to β-dystroglycan, which is part of a membrane bound dystrophin-associated glycoprotein complex (DGC) (fig. 1). B-dystroglycan is a transmembrane protein that is bound to the extracellular α-dystroglycan, which in turn is bound to laminin-2, a part of the extracellular matrix (ECM). The central rod domain can absorb mechanical force. Hereby the protein transmits energy produced by the actin-myosin contraction machinery via the cell membranes to the connective tissue and tendons surrounding the muscles, to maintain the energy-balance and prevent overstressing of the muscle fibres (Ehmsen et al., 2002).

phenotype have (nearly) normal life expectancies. Around 50% of patients also develops

The majority of female carriers shows no signs of disease. Only in 5 to 10% some degree of skeletal muscular weakness and enlarged calves are reported, but this is generally very mild and often does not affect daily activities. A small part of these carriers develops cardiomyopathy later in life; however most of the women with cardiac abnormalities on echocardiogram or ECG (left ventricular dilatation and decreased shortening fraction), are asymptomatic. There is no relation between the presence of skeletal muscle weakness and

At present there is no cure for DMD. However, during the past decades pharmacological interventions and improved care (e.g. physiotherapy and assisted ventilation) have led to increased function and quality of life and prolonged life expectancy for currently diagnosed patients into their forties. The current standard of care also consists of corticosteroids (mainly predniso(lo)ne or deflazacourt). These are anti-inflammatory/immunosuppressive drugs that have shown to improve muscle function, prolong ambulation for around 3 years

DMD is caused by a genetic defect in the *DMD* gene. In approximately 33% of cases this is a de novo (new) mutation. The *DMD* gene is located on the short arm of the X-chromosome (at Xp21). It is the largest gene in the human genome consisting of 2 220 223 base pairs. The coding sequence spans around 0.5% (11 058 bases) of the gene, dispersed over 79 exons. Mutations in the gene causing a disruption of the open reading frame or introducing a premature stop codon lead to a complete absence of a functional dystrophin protein. Dystrophin consists of 3 685 amino acids and has a molecular weight of 427 kDa (Muntoni et al., 2003). The protein is located inside the muscle fibres and forms a bridge between the actin cytoskeleton and the extracellular matrix (ECM). Thereby it provides mechanical stability to the muscle fibres during each contraction. The protein consists of 4 domains: first an N-terminus, containing 2 actin-binding domains (ABD), both consisting of a CH1-and a CH2-domain, which are bound to contractile structures (F-actin) inside the muscle cells. This is followed by a central domain, so called central rod domain, consisting of 24 spectrin-like triple helical coiled repeat units, interrupted by 4 proline-rich hinge regions. A third actinbinding domain is present between repeat 11 and 17 (Amann et al., 1998), while repeat 16-17 contain a binding site for neuronal nitric oxide synthase (nNOS) (Lai et al., 2009). Subsequently the protein contains a cysteine-rich part and finally a C-terminal domain. The cysteine-rich domain binds to β-dystroglycan, which is part of a membrane bound dystrophin-associated glycoprotein complex (DGC) (fig. 1). B-dystroglycan is a transmembrane protein that is bound to the extracellular α-dystroglycan, which in turn is bound to laminin-2, a part of the extracellular matrix (ECM). The central rod domain can absorb mechanical force. Hereby the protein transmits energy produced by the actin-myosin contraction machinery via the cell membranes to the connective tissue and tendons surrounding the muscles, to maintain the energy-balance and prevent overstressing of the

cardiomyopathy (Emery, 1993).

the development of cardiomyopathy (Grain et al., 2001).

**2.** *DMD* **gene and dystrophin protein** 

**2.1 Genetic defect in DMD** 

muscle fibres (Ehmsen et al., 2002).

and to have a positive effect on cardiac function (Bushby et al., 2010).

The dystrophin-associated glycoprotein complex (DGC) is composed of α- and β-dystroglycan, a sarcoglycan-sarcospan complex and the dystrophin containing cytoplasmic complex. Dystrophin (purple) forms the link between the actin cytoskeleton with its N-terminal domain and extracellular matrix component laminin-2 (lilac) via α- and β-dystroglycan (dark blue) with its C-terminal domain. Bdystroglycan is also bound to the sarcoglycan-sarcospan complex (light blue/black) and to caveolin-3 (orange), a scaffolding protein of skeletal muscle caveolae. Furthermore, the C-terminal domain of dystrophin is connected to α-dystrobrevin (green) and syntrophin (salmon pink), which recruits nNOS (yellow), a vasodilator, to the membrane. Α-dystrobrevin, in turn, is linked to syncoilin (brown), forming a bridge between the DGC and the desmin intermediate filament protein network (brown).

Fig. 1. The dystrophin-associated glycoprotein complex

In addition to its mechanical linker function, dystrophin is involved in the organisation of the DGC as well as many other proteins, the maintenance of the calcium homeostasis and control of the growth of the muscle cells (Hoffman et al., 1987). In the DGC, β-dystroglycan is connected to a complex of α-, β-, γ- and δ-sarcoglycans and sarcospan. This complex functions in maintaining membrane stability (Miller et al., 2007). B-dystroglycan is also bound to caveolin-3, a structural protein of skeletal muscle caveolae, small invaginations of the plasma membrane playing a role in, among others, signal transduction. Caveolins act as scaffolding proteins to compartmentalise and functionally regulate signalling molecules (Hezel et al., 2010). Furthermore, the C-terminal domain of dystrophin is connected to αdystrobrevin and syntrophin. nNOS is recruited to the membrane by binding to dystrophin and syntrophin. In contracting muscles, nNOS produces NO to induce vasodilatation in order to increase the local blood flow necessary for the increased mechanical load. The absence of nNOS in DMD causes abnormal vasoconstriction and ischemic stress, which contributes to the muscle degeneration (Brenman et al., 1995). Syntrophin is also connected to sodium channels, which are involved in regulating the Na+ distribution. In DMD, defects in cardiac conduction systems are thought to be caused by disturbances in Na+ distribution (Gee et al., 1998). A-dystrobrevin is linked to syncoilin too, thereby forming a bridge between the DGC and the desmin intermediate filament protein network at the neuromuscular junction (Newey et al., 2001).

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 59

In contrast to DMD, suffering from a complete absence dystrophin, in BMD a shorter, but partly functional, dystrophin protein is present. This discrepancy can be explained by the type of mutation that affects the *DMD* gene. In DMD mutations cause a disruption of the open reading frame or a premature stop codon, whereby the transcription of the gene stops prematurely and no functional protein is formed. In BMD the open reading frame stays intact (i.e. the size of the deletion in base pairs is divisible by 3), thereby translation can continue and a shorter protein is formed (fig. 2). This reading frame rule holds for over 90% of the cases (Aartsma-Rus et al., 2006b; Koenig et al., 1989). Only in-frame deletions that are very large (>36 exons) or deleting essential parts of the protein (the complete actin-binding domain or (part of) the cysteine-rich domain) lead to DMD. Furthermore, a small number of mutations that do disrupt the reading frame, lead to BMD instead of DMD (2%). This is probably due to correction of the reading frame at RNA level (Aartsma-Rus et al., 2006b).

The most widely used model for DMD is the *mdx* mouse model (C57Bl/10ScSn-*DMDmdx/J*). These mice have a single base substitution within exon 23, leading to a premature stop codon, so a truncated, non-functional dystrophin protein is formed (Sicinski et al., 1989). Despite the absence of dystrophin, the phenotype of the *mdx* mice is relatively mild compared to human DMD patients. However, compared to wild-type mice, their muscles are clearly dystrophic and functionally impaired (Chamberlain et al., 2007). Nevertheless, their life span is only slightly reduced and the muscular weakness is mild. This is probably due to compensatory mechanisms, like the upregulation of utrophin, a dystrophin homologue, which can partly take over its function. Mice that lack both dystrophin and utrophin (*mdx/utrn-/-*, double knock-out mice) show a very severe, progressive muscular dystrophy. Their muscles display several signs of damage and are rapidly replaced by fibrotic and adipose tissue. Furthermore, these mice are functionally impaired, have an arched spine and a life span of 20 weeks at maximum (Deconinck et al., 1997). Due to the very severe phenotype and short life span, *mdx/utrn-/-* mice are not practical as experimental model. An intermediate model is the *mdx* mouse with haploinsufficiency for utrophin (*mdx/utrn+/-*). Inflammation and fibrosis in both skeletal muscle and diaphragm are more severe than in the *mdx* mouse, but less than in the *mdx/utrn-/-* mouse. Their life span is

Next to the naturally occurring mutation in the *mdx* mouse, several DMD mutations have been induced in mice. For example, treatment of mice with the chemical *N*-ethylnitrosourea (ENU), a powerful mutagen in mice, resulted in several new *mdx*-like mouse models (B6Ros.Cg-Dmd*mdx*–*Cv*/J). *Mdx2Cv* has a mutation in a splice site in exon 43 (causing alternative splicing, resulting in out-of-frame transcripts), *mdx3Cv* a mutation in intron 65 (inducing a new splice site, resulting in out-of-frame transcripts), *mdx4Cv* a mutation in exon 53 (premature stop codon) and *mdx5Cv* a mutation in exon 10 (frame-shift by introduction of a new splice site). All these mice have a phenotype comparable to the *mdx* mouse (Chapman et al., 1989). In addition, several mouse models have been generated that only affect 1 or a

significantly longer than that of *mdx/utrn-/-* mice (Zhou et al., 2008).

few of the different dystrophin isoforms.

**2.2 Genetic defect in BMD** 

**2.3 Animal models for DMD 2.3.1 Mouse models for DMD** 

Furthermore, in addition to the most common form of the dystrophin protein found in muscles, additional full-length and shorter isoforms of dystrophin exist. This is due to the presence of at least 7 different promoters and alternative splicing events. Three full-length variants exist (including the muscle isoform), which only differ in their first exon. In addition to the muscle promoter expressed in skeletal muscle and cardiomyocytes, a brain promoter drives expression in the cortical neurons and hippocampus of the brain and a Purkinje promoter in the cerebellar Purkinje cells. Four internal promoters lead to the production of shorter dystrophin proteins, lacking the actin-binding domains, expressed in specific tissues. In addition, alternative splicing facilitates the expression of many more dystrophins with a tissue-specific function (Muntoni et al., 2003).

a.) In the normal situation pre-mRNA is spliced to produce mRNA, which in turn is translated into the dystrophin protein. This fully functional protein forms a bridge between the actin cytoskeleton and the extracellular matrix. b.) In DMD mutations lead to a disruption of the open reading frame and translation into protein stops prematurely. A truncated, non-functional dystrophin protein (which is degraded) is formed and the bridge function is lost. c.) In BMD mutations do not disrupt the open reading frame and translation into a shorter, but largely functional protein can occur. The bridge function is maintained.

Fig. 2. The reading frame rule

#### **2.2 Genetic defect in BMD**

58 Neuromuscular Disorders

Furthermore, in addition to the most common form of the dystrophin protein found in muscles, additional full-length and shorter isoforms of dystrophin exist. This is due to the presence of at least 7 different promoters and alternative splicing events. Three full-length variants exist (including the muscle isoform), which only differ in their first exon. In addition to the muscle promoter expressed in skeletal muscle and cardiomyocytes, a brain promoter drives expression in the cortical neurons and hippocampus of the brain and a Purkinje promoter in the cerebellar Purkinje cells. Four internal promoters lead to the production of shorter dystrophin proteins, lacking the actin-binding domains, expressed in specific tissues. In addition, alternative splicing facilitates the expression of many more

a.) In the normal situation pre-mRNA is spliced to produce mRNA, which in turn is translated into the dystrophin protein. This fully functional protein forms a bridge between the actin cytoskeleton and the

extracellular matrix. b.) In DMD mutations lead to a disruption of the open reading frame and translation into protein stops prematurely. A truncated, non-functional dystrophin protein (which is degraded) is formed and the bridge function is lost. c.) In BMD mutations do not disrupt the open reading frame and translation into a shorter, but largely functional protein can occur. The bridge

function is maintained.

Fig. 2. The reading frame rule

dystrophins with a tissue-specific function (Muntoni et al., 2003).

In contrast to DMD, suffering from a complete absence dystrophin, in BMD a shorter, but partly functional, dystrophin protein is present. This discrepancy can be explained by the type of mutation that affects the *DMD* gene. In DMD mutations cause a disruption of the open reading frame or a premature stop codon, whereby the transcription of the gene stops prematurely and no functional protein is formed. In BMD the open reading frame stays intact (i.e. the size of the deletion in base pairs is divisible by 3), thereby translation can continue and a shorter protein is formed (fig. 2). This reading frame rule holds for over 90% of the cases (Aartsma-Rus et al., 2006b; Koenig et al., 1989). Only in-frame deletions that are very large (>36 exons) or deleting essential parts of the protein (the complete actin-binding domain or (part of) the cysteine-rich domain) lead to DMD. Furthermore, a small number of mutations that do disrupt the reading frame, lead to BMD instead of DMD (2%). This is probably due to correction of the reading frame at RNA level (Aartsma-Rus et al., 2006b).

#### **2.3 Animal models for DMD**

#### **2.3.1 Mouse models for DMD**

The most widely used model for DMD is the *mdx* mouse model (C57Bl/10ScSn-*DMDmdx/J*). These mice have a single base substitution within exon 23, leading to a premature stop codon, so a truncated, non-functional dystrophin protein is formed (Sicinski et al., 1989). Despite the absence of dystrophin, the phenotype of the *mdx* mice is relatively mild compared to human DMD patients. However, compared to wild-type mice, their muscles are clearly dystrophic and functionally impaired (Chamberlain et al., 2007). Nevertheless, their life span is only slightly reduced and the muscular weakness is mild. This is probably due to compensatory mechanisms, like the upregulation of utrophin, a dystrophin homologue, which can partly take over its function. Mice that lack both dystrophin and utrophin (*mdx/utrn-/-*, double knock-out mice) show a very severe, progressive muscular dystrophy. Their muscles display several signs of damage and are rapidly replaced by fibrotic and adipose tissue. Furthermore, these mice are functionally impaired, have an arched spine and a life span of 20 weeks at maximum (Deconinck et al., 1997). Due to the very severe phenotype and short life span, *mdx/utrn-/-* mice are not practical as experimental model. An intermediate model is the *mdx* mouse with haploinsufficiency for utrophin (*mdx/utrn+/-*). Inflammation and fibrosis in both skeletal muscle and diaphragm are more severe than in the *mdx* mouse, but less than in the *mdx/utrn-/-* mouse. Their life span is significantly longer than that of *mdx/utrn-/-* mice (Zhou et al., 2008).

Next to the naturally occurring mutation in the *mdx* mouse, several DMD mutations have been induced in mice. For example, treatment of mice with the chemical *N*-ethylnitrosourea (ENU), a powerful mutagen in mice, resulted in several new *mdx*-like mouse models (B6Ros.Cg-Dmd*mdx*–*Cv*/J). *Mdx2Cv* has a mutation in a splice site in exon 43 (causing alternative splicing, resulting in out-of-frame transcripts), *mdx3Cv* a mutation in intron 65 (inducing a new splice site, resulting in out-of-frame transcripts), *mdx4Cv* a mutation in exon 53 (premature stop codon) and *mdx5Cv* a mutation in exon 10 (frame-shift by introduction of a new splice site). All these mice have a phenotype comparable to the *mdx* mouse (Chapman et al., 1989). In addition, several mouse models have been generated that only affect 1 or a few of the different dystrophin isoforms.

AON-Mediated Exon Skipping for Duchenne Muscular Dystrophy 61

patients rare, dystrophin-positive (so-called "revertant" fibres) were found, which are the result of spontaneous exon skipping or secondary mutations restoring the reading frame in these fibres and allowing dystrophin production. Therefore it was hypothesised that using AONs to induces skipping of specific exons could lead to the restoration of the reading frame and thereby production of slightly shorter dystrophin proteins, as found in BMD and revertant fibres (fig. 3) (van Ommen et al., 2008). This approach is mutation-specific and a large variety in mutations exists among DMD patients. Fortunately, 2 "hotspots" (a major around exon 43 to 53 and a minor spanning exons 2 to 20) exist, comprising a large proportion of the mutations (Aartsma-Rus et al., 2006b). In this Chapter we will describe the development of this therapeutic approach. We are aware that many excellent papers about exon skipping for DMD exist. Due to space constraints it was not feasible to cover them all.

For a recent overview see Aartsma-Rus, *RNA Biology* 2010 (Aartsma-Rus, 2010).

In DMD mutations in the *DMD* gene lead to a disruption of the open reading frame (in this example a deletion of exon 50), thereby preventing production of a functional dystrophin protein. Binding of an exon-specific AON (in this example against exon 51) hides the exon from the splicing machinery. The exon will be 'skipped' and not incorporated in the mRNA. Thereby the reading frame is restored and translation of a shorter, but still largely functional dystrophin protein can occur, which is similar to the

To prevent activation of RNase H the 2'-*O* position of the ribose was modified (2'-*O*-methyl (2OME) or 2'-*O*-methoxyethyl (2OMOE)). Furthermore, various chemical modifications (fig. 4) have been developed, which differ in sugar and backbone chemistry and have different

proteins found in BMD.

**3.2 Backbone chemistries** 

Fig. 3. Antisense oligonucleotide-mediated exon skipping

#### **2.3.2 Canine models for DMD**

The Golden retriever muscular dystrophy (GRMD) dog is a spontaneously occurring canine model for Duchenne muscular dystrophy. These dogs have a single base substitution in the 3' consensus splice site of intron 6, resulting in skipping of exon 7, thereby introducing a premature stop codon in exon 8. The course of the disease is more comparable to human patients than that of the *mdx* mouse. The dogs display rapid and fatal muscular dystrophy, characterised by muscle atrophy, myofibre degeneration, replacement by fibrotic and adipose tissue and cardiomyopathy (Sharp et al., 1992). Most affected animals die within a few years, mainly due to degeneration of the cardiac muscle (Howell et al., 1997). Although phenotypically the GRMD dog seems a better model for DMD, it shows a lot of interindividual variation in the severity of the pathology. Some animals die within days after birth, whereas others appear almost normal and live for years (Ambrosio et al., 2008). This makes the dogs less suitable for experimental use, due to standardisation problems.

Because of the large size of the golden retriever, the GRMD dog has been bred with a much smaller beagle to generate the canine X-linked muscular dystrophy (CXMDj) model. These dogs have a milder phenotype compared to GRMD dogs and therefore have a longer life span (Shimatsu et al., 2003).

In addition to the above mentioned large phenotypical variation, experiments with dogs are very costly. Dogs have a long breeding time and the availability is low (a heterozygous breeding program is needed, due to the severity of the phenotype). Furthermore, for therapeutic studies the size of the dogs requires large amounts of compound.
