Exp Physiol 2011 Fairclough Expphysiol.2010.053025

8/11/2019 Exp Physiol 2011 Fairclough Expphysiol.2010.053025

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Progress in therapy for Duchenne muscular dystrophy

Rebecca J. Fairclough*, Akshay Bareja*, Kay E. Davies#

MRC Functional Genomics Unit, Department of Physiology Anatomy and Genetics, Universityof Oxford OX1 3PT, UK

*equal contribution

# corresponding author

)by guest on September 9, 2014ep.physoc.orgDownloaded from Exp Physiol (
http://ep.physoc.org/http://ep.physoc.org/http://ep.physoc.org/http://ep.physoc.org/


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Abstract

Duchenne muscular dystrophy is a devastating muscular dystrophy of childhood.

Mutations in the dystrophin gene destroy the link between the internal muscle filaments

and the extracellular matrix resulting in severe muscle weakness and progressive muscle

wasting. There is currently no cure, and, whilst palliative treatment has improved, affected

boys are normally in a wheelchair by twelve years of age and die from respiratory or

cardiac complications in their 20s-30s. Therapies currently being developed include

mutation-specific treatments, DNA- and cell-based therapies, and drugs which aim to

modulate cellular pathways or gene expression. This review aims to provide an overview

of the different therapeutic approaches aimed at reconstructing the dystrophinassociated

protein complex, including restoration of dystrophin expression and upregulation of the

functional homologue utrophin.



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Introduction

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease that afflicts one in

every 3,500 males (Emery, 1992). It is caused by the absence of the protein

dystrophin(Koenig et al., 1987). DMD patients are generally boys who exhibit early

symptoms (around 3-5 years of age) of abnormal gait, calf muscle pseudohypertrophy

(Emery, 1992) and the characteristic Gowers sign (weakness of the proximal muscles,

particularly those of the lower limb; Gowers, 1985). Respiratory complications, including

sleep-disordered breathing and apnoea, arise during the teenage years resulting in

headaches, nausea, fatigue and poor appetite (Emery, 1992; Cox & Kunkel, 1997). Patients

are usually wheelchair-bound by 12 years of age (Emery, 1992). Dilated cardiomyopathy

of varying severity is evident from abnormal ECGs in most patients by 18 years of age, and

is currently responsible for the death of approximately 10-40% of all DMD patients(Baxter, 2006). The development of scoliosis is coincident with permanent wheelchair use,

rapidly progressing during puberty and adversely affecting respiratory function (Emery &

Muntoni, 2003). Many patients die of respiratory failure in their twenties (Emery &

Muntoni, 2003). Becker muscular dystrophy (BMD) is a milder disease caused by the

reduced expression of dystrophin or expression of a partially functional protein. Some

BMD patients only present with a cardiomyopathy and remain ambulant into their fifties or

sixties (Yazaki et al., 1999). There is currently no cure for DMD. Prednisolone has been

shown to increase the ambulatory period for some patients (Connolly et al., 2002) but more

effective interventions are desperately needed.

Dystrophin and the Dystrophin Associated Protein Complex

Dystrophin is a 427-kDa protein that is comprised of four domains: the N-terminal domain

(which binds actin), the rod domain (which consists of 26 spectrin-like repeats), a cysteine-

rich domain and a C-terminal domain (Koenig et al., 1988).

Skeletal muscle is made up of thousands of muscle fibres (myofibres), each of which

is surrounded by a plasma membrane (sarcolemma) and an overlying basal lamina.

Dystrophin is expressed in skeletal muscle at the sarcolemma and is enriched at the

myotendinous junction (MTJ; the junction of muscle fibres and tendons) and the



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neuromuscular junction (NMJ; the junction of a motor neuron with a muscle fibre)

(Khurana et al., 1991). Dystrophin binds to filamentous (F)-actin (a constituent member of

the cytoskeleton) via its N-terminus and a cluster of basic repeats 11-17 in its rod domain

(Amann et al., 1999). The protein binds dystroglycan via its cysteine-rich domain, and

-

dystrobrevin and

-syntrophin via its C-terminal domain (Junget al., 1995; Yanget al.,

1995; Sadoulet-Puccioet al., 1997). Figure 1 (taken from Davies & Nowak, 2006)

illustrates the mechanical link dystrophin creates between the internal cytoskeleton and the

external extracellular matrix.

The main function of dystrophin is to stabilize the sarcolemma. Due to the elasticity

and flexibility of its large central rod domain consisting of triple helical repeats, dystrophin

has been proposed to protect the myofibre from contraction-induced stress (Petrof et al.,

1993). It has also been shown to form a mechanically strong link between the sarcolemma

and the internal cytoskeleton via its interaction with gamma-actin filaments (Rybakova et

al., 2000). It has also been postulated to play a role in signalling pathways, and this is

discussed further below.

Preclinical animal models of DMD

The most widely used DMD mouse model is the mdx mouse, which is a naturally occurring

mutant that has a premature stop codon in exon 23 of its dystrophin gene due to a point

mutation, resulting in the complete absence of full-length dystrophin expression. Muscle

degeneration follows a characteristic progression in the mdx mouse, beginning with a sharp

increase in myofibre necrosis and elevated serum creatine kinase levels around 3 weeks of

age (McGeachie et al., 1993), which decreases in severity to a chronic low level of damage

by 8 weeks of age (Spencer & Tidball, 1996). This persists until about 1 year of age

following which there is a further reduction in the levels degeneration. Despite being the

exact murine analogue of human DMD patients, mdx mice do not display a disease

phenotype that is as severe as that of DMD patients. For example, they do not show any

impairment in mobility throughout their lifetime and, with the exception of the diaphragm,



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their skeletal muscles do not display similar levels of fibrosis (Stedmanet al., 1991;

Connollyet al., 2001; Rollandet al., 2006)

Due to the relatively mild phenotype of the mdxmouse, many attempts have been made to

exacerbate this phenotype by generating double knockouts. These have been reviewed in detail

elsewhere (Willmann et al., 2009). The double knockout that has been used most extensively in

preclinical studies for DMD is the utrophin/dystrophin double knockout (dKO, mdx; utrn-/-

)

which was generated by crossing mdxmice with a knock-out of the functional dystrophin

homologue, utrophin (Deconinck et al., 1997; Grady et al., 1997; the role of utrophin is described

in detail below). The dKO mouse displays a disease phenotype far more similar in its severity

to that of DMD patients. Unlike mdxmice, dKO mice have a significantly reduced lifespan of 4-

20 weeks, and display many of the hallmark symptoms of DMD, such as severe muscle

weakness, joint contractures, kyphosis (curvature of the upper spine) and pronounced growth

retardation post-weaning. The accurate recapitulation of the dystrophic progression of DMD in

this mouse model, coupled with the absence of the compensatory power of utrophin, means that

the dKO mouse can serve as a better model than the mdxmouse in which to test the effectiveness

of therapeutic strategies. For example, this model has recently been used to test the effectiveness

of an exon-skipping strategy (Goyenvalle et al., 2010).

The best characterized and most widely used canine model of DMD is the Golden

retriever (GRMD) dog model. GRMD lacks dystrophin due to incorrect splicing resulting in a

truncated transcript (Sharp et al., 1989). These dogs display a phenotype that is very similar to

human DMD patients. The GRMD dog has been successfully used to test the therapeutic

potential of mesoangioblast administration (Sampaolesi et al., 2006). The major problem with

the use of these dogs is that they display a high degree of phenotypic variation (Ambrosio et

al., 2008).

Pathophysiological consequences of dystrophin absence

Since the DAPC plays many roles in the proper functioning of the skeletal muscle, the

absence of dystrophin, and hence the impaired recruitment of the members of the DAPC to

the sarcolemma, creates a pathophysiological cascade with numerous adverse downstream



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effects. Since the primary function of dystrophin is to stabilize the sarcolemma, its absence

renders the sarcolemma vulnerable to damage-inducing factors (Petrof et al., 1993). As the

affected organism grows in size, increased amounts of stress will be imposed on the

sarcolemma as a result of increased load and force generation. The resulting membrane

instability has many deleterious consequences. Transient tears in the membrane promote

the unregulated influx of Ca2+

ions into the sarcoplasm which is thought to have toxic

effects primarily via its effect on mitochondrial genes (Chen et al., 2000). The continuous

degeneration and regeneration of muscle tissue as a result of stress factors has been

suggested to be responsible for elevated levels of fibrosis as exemplified by excess collagen

deposition (Morrison et al., 2000). The lack of dystrophin and, hence,

-syntrophin, results

in the down regulation and relocalization of nNOS at the sarcolemma (Chang et al., 1996).

nNOS activity is thought to counter the vasoconstrictive response of the muscle,

particularly during periods of exercise (Thomaset al., 1998; Laiet al., 2009a). The

dysregulation of blood flow as a result of its absence is therefore thought to result in

functional ischaemia (Sander et al., 2000). One study suggests that the absence of

dystrophin results in the downregulation of Akt, which normally functions to inhibit

MuRF1 and MAFbx: E3 ubiquitin ligases that are involved in muscle atrophy by targeting

components of the myofibre for proteasomal degradation (Acharyya et al., 2005). This

study also showed that loss of dystrophin expression led to aberrant glycosylation of

-

dystroglycan and

-sarcoglycan, both of which are key members of the DAPC.

Therapies for DMD

Numerous different therapeutic strategies have been devised to correct the various

deleterious consequences of dystrophin absence. This review covers some of the most

promising approaches to therapy which tackle the primary cause. Treatments which

alleviate the secondary effects of the disease have been reviewed elsewhere (Zhou & Lu;Bogdanovichet al., 2004; Bushbyet al., 2009; Zhou & Lu, 2010). These strategies can be

divided into four categories: cell therapy (myoblast transplantation and stem cell therapy),

gene replacement therapy, mutation-specific approaches (exon skipping and suppression of

premature termination codons) and utrophin upregulation.



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Cell therapy

Cell therapy involves the delivery of cells that make new muscle to diseased areas. These

can either be muscle precursor cells or stem cells that have the ability to differentiate into

muscle cells. The cell therapy strategy that has experienced the most success so far has

been the transplantation of myoblasts into diseased tissue.

Myoblasts are muscle precursor cells that can differentiate to form myofibres.

Myoblast transplantation (MT) involves the derivation of myoblast cells from healthy

donor skeletal muscle, expansion of these cells in culture, and administration to dystrophic

tissue. The incorporation of donor myoblasts into recipient myofibres results in a

phenomenon known as gene complementation, which means the expression of both

exogenous and host genes in the myofibre syncitium (Watt et al., 1982). Following good

transplantation results in immunosuppressed mdxmice (Partridgeet al., 1989; Morgan &

Partridge, 1992; Vilquinet al., 1994) great expectation saw this therapy propelled into

human trials. Sadly, these trials yielded only limited positive results, with a maximum of

10 % donor dystrophin-positive fibres in one out of twelve patients (Mendell et al., 1995).

This poor result has been attributed to poor immunosuppression combined with insufficient

numbers of transplanted cells and insufficient distribution of cells (Gussoniet al., 1992;

Karpatiet al., 1993; Tremblayet al., 1993a; Tremblayet al., 1993b; Mendellet al., 1995).

Since then improvements in tissue repopulation were achieved in DMD animal models

through the use of muscle slice grafts (Fan et al., 1996), although translation into clinical

trials would of course be limited to larger more surgically accessible dystrophic muscles. It

has become clear that the success of MT transplantation is also closely related to the choice

of immunosuppressive drug used (Hong et al., 2002). Current hopes are pinned on the use

of donor genetically modified myoblasts to reduce the patient immune response (Li et al.,

2005; Benchaouiret al., 2007; Kazukiet al., 2010). Lots of studies have been performed to

identify factors that may improve myoblast migration through adult tissues (Fakhfakhetal.; Gerardet al.; Smythe & Grounds, 2000; Smytheet al., 2001; Smythe & Grounds,

2001). Other studies suggest increasing the number of cells transplanted may not be

beneficial (Pellegrini & Beilharz); moreover, a recent study by Hall et al. (Hall et al.)

suggested that only a very small number of satellite cells are able to repopulate muscle

successfully. This study was performed using a small number of satellite cells that were



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cultured in vitro and subsequently transplanted into muscle whilst still attached to the

myofibres. Perhaps the microenvironment of the myofibre provides novel elements that are

crucial for the subsequent survival and proliferation of the satellite cells following

transplantation?

It was initially thought that only satellite cells were responsible for the growth and

maintenance of skeletal muscle. There are, however, other cells found in skeletal muscle

that have myogenic potential, namely muscle-derived stem cells (MDSCs), muscle side-

population (mSP) cells and muscle-derived CD133+ progenitors. Certain stem cells derived

from non-muscle have also been shown to be able to participate in myogenesis. For

example, bone marrow-derived mesenchymal stem cells (MSCs) can differentiate into

mesodermal cells, including myoblasts (Bhagavati & Xu, 2004). MSCs have the

advantages of being able to fuse with and genetically complement dystrophic muscle,

possessing anti-inflammatory properties, and producing factors that enhance the activity of

endogenous repair cells (Ichim et al., 2010). However, most of the studies using MSCs in

animal models did not show an appreciable improvement in muscle contractile force (Ichim

et al., 2010). Another type of stem cell whose use has been proposed to be a possible

therapeutic option is the mesoangioblast cell. This is a multipotent progenitor of

mesodermal tissue whose transplantation in dystrophic dogs has been shown to result in the

re-expression of dystrophin in myofibres and an improvement in muscle contractile force

(Sampaolesi et al., 2006).

A major limitation that needs to be overcome if cell therapy is going to be used to

successfully treat DMD patients is the inability of intravenous and intraperitoneal injections

of myoblasts to repopulate dystrophic muscle (Partridge, 1991), necessitating the use of

intramuscular injections. The major disadvantage of this delivery method is that the

myoblasts fuse mainly with the myofibres that neighbor the site of injection, resulting in

restricted zones of muscle growth and recovery (El Fahimeet al., 2000; Torrenteet al.,

2000; El Fahimeet al., 2002; Skuket al., 2002). Of course, local transfer also makes

treatment of less accessible muscles, such as the diaphragm, virtually impossible.

Stem cell therapy in clinical trial for DMD includes CD133+ (Torrente et al., 2007)

and mesoangioblasts (in progress; under the direction of Giulio Cossu).



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Conventional gene replacement strategies

Over recent years there has been significant advancement in research towards an effective

therapy for DMD using direct gene replacement approaches. This strategy is limited by the

large size (14kb) of the dystrophin gene. However, there are very mildly affected BMD

patients who are ambulant into their 50s and 60s who lack up to 50% of the gene sequence.

Significantly, these deletions occur in the rod domain of the protein encoding spectrin

repeats (Englandet al., 1990; Loveet al., 1991; Matsumuraet al., 1994). These gene

deletions have formed the basis of the generation of mini- and micro-dystrophin genes with

increasingly large sections of the rod domain removed to enable them to be cloned into

AAV vectors (for a detailed review see (Goyenvalle & Davies, 2011a)). Systemic delivery

of mini-dystrophin genes with AAV9 has been very effective in mouse and dog (Pichavant

et al., 2010). However, a recent clinical trial using intramuscular injections of AAV2 into

DMD patients did not result in good restoration of dystrophin expression and highlighted

the potential for T cell immunity to dystrophin epitopes (Mendellet al., 2010). An

alternative strategy is the delivery of micro- genes encoding the dystrophin-related protein

utrophin (see below) which should not elicit an immune response.

Problems faced by this type of approach to therapy include the production of

sufficient quantities of virus and circumventing the immune response encountered through

the introduction of a previously absent protein. Additionally, these shortened genes might

not be capable of completely rescuing the DAPC defect because their protein products will

be unlikely to recapitulate all of dystrophins interactions with members of the DAPC. For

example, full length dystrophin (together with syntrophin) recruits neuronal nitric oxide

synthase (nNOS) to the sarcolemma (Hillieret al., 1999; Kameyaet al., 1999; Tochioet

al., 1999; Adamset al., 2000; Laiet al., 2009a). nNOS localized at the membrane

counteracts

-adrenergic vasoconstriction during muscle contraction by allowing diffusion

of nitric oxide. Failure to do this aggravates muscle disease by resulting in functional

ischaemia (Sander et al., 2000). It has been shown that the sarcolemmal targeting of nNOS

is dependent on the spectrin-like repeats 16 and 17 of the rod domain (Lai et al., 2009b),

which means that any truncated dystrophin transgene that lacks this region is not likely to

be an entirely functional replacement.



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A phase I safety study of mini-dystrophin is currently being performed under the

direction of Asklepios Biopharmaceutical Inc. and Jerry Mendell.

Suppression of premature termination codons

Studies have shown that mammalian cells treated with high concentrations of

aminoglycoside antibiotics like gentamicin promote readthrough of premature stop codons

by interfering with the ribosomes ability to recognize these stop signals (Manuvakhova et

al., 2000). Gentamicin treatment could therefore be applied to DMD patients with nonsense

mutations in their dystrophin gene. A six-month trial of gentamicin on DMD patients

resulted in a significant increase in dystrophin expression in skeletal muscle (with the

highest level approaching 13-15% of normal expression in one individual) and decreased

levels of creatine kinase - a marker of muscle damage. However, one of the individuals

developed an immune response to the newly-expressed dystrophin protein (Malik et al.,

2010). Additional problems with gentamicin include its lack of potency and possible toxic

effects (Welch et al., 2007).

PTC124 is a synthetic drug that has been shown to have the ability to induce

readthrough of premature but not normal stop codons. Treatment of mdx mice with

PTC124 resulted in a significant increase in dystrophin expression in skeletal muscle and

partial (yet significant) recovery of muscle contractile force (Welch et al., 2007). Some of

the results from this study have however been recently called into question and a Phase IIb

clinical trial of PTC124 has recently been suspended (Finkel, 2010; Guglieri and Bushby,

2010). It is estimated that 13 % of DMD patients have nonsense mutations and could

therefore be treated by nonsense suppression.

Exon skipping

Exon skipping is a promising RNA based approach which could target up to 83% of DMD

patients with deletions (Aartsma-Ruset al., 2009). It relies on synthesised antisense

oligonucleotides (AONs) carried into the cell using a range of different chemical



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backbones. These include peptide nucleic acids (PNA), 2-O-methyl-phosphorothiate-

AONs (2OMeAO), phosphorodiamidate morpholino oligomer (PMO) and cell penetrating

peptide-conjugated PMO (PPMO). AOs alter splicing by either sterically blocking splice

enhancer sequences or by altering secondary mRNA structure folding. Success with this

technique has been achieved in cell culture, and trials in mdx (Alter et al., 2006) and dKO

(Goyenvalle et al., 2010) have ameliorated the disease phenotypes of both mouse models.

Exon 51 skipping represents the therapy with the potential to target the largest percentage

(13 %) of DMD patients and, for this reason, clinical trials are currently concentrating on

this target. A Phase III systemic trial of GSK2402968 (GSK; originally developed by

Prosensa, Netherlands and called PRO51), based on the 2OMeAO chemistry and targeting

exon 51, is ongoing. A phase I/II clinical trial of AVI-4658 (AVI Biopharma, USA), again

targeting exon 51 and based on the PMO backbone, has recently been completed. Both

have demonstrated good tolerability of the drug and restoration of dystrophin expression at

the sarcolemma (Goemanset al., 2010; Shrewsburyet al., 2010). However, poor cellular

uptake, relative rapid clearance from the circulation, variable skipping efficiency between

muscles and poor efficacy in the heart are just several of the challenges standing between

these drugs and routine clinical use (for detailed review see (Goyenvalle & Davies,

2011a)). Furthermore, there are indications from preclinical trials that doses required for

functional improvement may be too high to make lifelong treatment economically possible

for the majority of patients. In view of this, much hope is being placed on the development

of PPMOs which demonstrate enhanced internalisation by muscle cells (Moulton &

Moulton, 2010) at much lower doses (Jearawiriyapaisarn et al., 2008; Yin et al., 2008; Yin

et al., 2009) and demonstrate efficacy in the heart (Jearawiriyapaisarn et al., 2010; Wu et

al., 2010; Wu et al., 2008; Yin et al., 2008b).

A novel approach to exon skipping in DMD therapy which is being further

developed in our laboratory by Aurelie Goyenvalle uses U7, a non spliceosome smallnuclear RNA (snRNA), normally involved in the processing of the histone mRNA 3 end,

to enhance the delivery of antisense sequences (Goyenvalle & Davies, 2011b). By slightly

modifying the binding site for Sm/Lsm proteins, U7 can be converted into a versatile tool

for splicing modulation. Delivery of the appropriately modified U7 snRNA using an

adeno-associated virus has demonstrated widespread dystrophin restoration in both the mdx



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mouse (Goyenvalleet al., 2004) and the GRMD (Vulin et al., 2011) models of DMD

following only a single dose. Through vectorizing the delivery of AONs, the resulting long

term restoration of dystrophin has obvious advantages over the need for repeated dosing

following administration of synthetic AONs.

Exon skipping holds a lot of promise but many hurdles remain. The fact that specific

oligonucleotide sequences will be required to target each individual exon that needs to be

skipped means that the regulatory authorities may consider each individual AON as a new

drug. There will be a need to target all muscle types, including the heart, as a minimum of

20 % dystrophin restoration has been postulated to be required for clinical efficacy

(Chamberlain, 1997). Repeated administration of the AOs may be required because of their

short half-life in muscle tissue, although this is not true for PMOs (for extensive review see

(Manzur & Muntoni, 2009)). Multi-exon skipping, which involves the exclusion of

multiple contiguous exons, has been proposed to be a way to circumvent the regulatory

constraints of individual drugs for individual exons by allowing the administration of the

same AO to patients with different mutational defects (Goyenvalle & Davies, 2011b).

Pharmacological upregulation of the dystrophin related protein, utrophin

Utrophin is an autosomal protein encoded by a gene on chromosome 6 in humans. The full-

length protein has 3,433 amino acids with a predicted molecular mass of 395 kDa

(compared to 3,678 amino acids and 427 kDa for dystrophin). The primary structure of

utrophin is very similar to that of dystrophin, with the N-terminal, cysteine-rich and C-

terminal domains displaying significant structural similarity, being 80 % identical to each

other (Tinsley et al., 1992). Unlike dystrophin which is expressed in muscle with lower

levels in brain, utrophin is ubiquitously expressed. Unlike dystrophin, which is expressed

throughout the sarcolemma of adult skeletal muscle cells, utrophin is only expressed at the

NMJ and MTJ, although it is found at the sarcolemma in early human development (Clerk

et al., 1993; Tome et al., 1994). Utrophin is also expressed across the entire sarcolemma in

developing and regenerating muscle (Khuranaet al., 1991; Tinsleyet al., 1992; Clerket

al., 1993; Ponset al., 1993) and in the skeletal muscle of mdxmice and DMD patients



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(Mizuno et al., 1993). Importantly, a significant inverse correlation between utrophin

expression and disease severity in DMD has been found (Kleopa et al., 2006).

Utrophin shares many of the same binding partners as dystrophin. The C-terminus

of utrophin has been shown to bind to members of the DAPC, such as

-dystrobrevin-1

(Peters et al., 1998) and

-dystroglycan (Ishikawa-Sakurai et al., 2004). Whilst both

utrophin and dystrophin bind

-dystroglycan with similar affinity through their ZZ domain,

their modes of binding slightly differ (Ishikawa-Sakurai et al., 2004). Also, like

dystrophin, utrophin binds to cytoskeletal F-actin. However, again, as in the case of

-

dystroglycan binding, the mode of contact is different. Whilst dystrophin binding occurs

through two independent actin-binding sites, utrophin binding occurs through one

continuous stretch of the protein (Amannet al., 1998; Rybakova & Ervasti, 2005).

Mechanistically, it has been postulated that this difference enables dystrophin to act as a

molecular shock absorber to dampen elastic recoil during contraction or stretch. In

contrast, utrophin may normally function as a molecular ruler to help define the length of

costameric actin filaments during muscle development. The strong similarities between

dystrophin and utrophin prompted the hypothesis that utrophin upregulation could lead to

the functional replacement of dystrophin at the sarcolemma. Over expression of minigenes

from an utrophin transgene driven by the human skeletal actin promoter in the mdxmouse

has been shown to prevent the dystrophic pathology (Tinsleyet al., 1996; Gilbertet al.,

1999). This model was named the Fiona mouse and is a transgenic mdxmouse that

expresses full-length utrophin in skeletal muscle at a 3-4 fold higher level than in non-

transgenic mdxmouse muscle. These mice are phenotypically indistinguishable from wild-

type mice in sedentary conditions and show complete correction of the dystrophic

phenotype, displaying even myofibre size, normal levels of muscle degeneration and

regeneration, and similar levels of force production by the extensor digitorum longus

(EDL) muscle compared to wild-type muscle (Tinsley et al., 1998). Whilst Li et al. (Li etal., 2010) recently demonstrated that utrophin does not completely compensate in exercised

mice, critically, it does stabilise the sarcolemma (Bareja, 2011). Delivery of utrophin using

intramuscular injections of adenoviruses in the GRMD also showed an improvement of

phenotype (Cerletti et al., 2003). In the mdxmouse utrophin expression initiated earlier in

development has a more potent therapeutic effect than when initiated later, exactly as is



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observed for dystrophin (Liu et al., 2005; Squire et al., 2002). Crucially for drug

development, body-wide overexpression of utrophin at levels which prevent the pathology

does not have detrimental effects in the mdxmouse (Fisher et al., 2001). These studies

collectively indicate that upregulating utrophin could have a powerful therapeutic effect in

DMD patients.

Utrophin expression is predominantly driven by two promoters: A and B (Burton et

al., 1999). The utrophin A promoter is responsible for the skeletal muscle-specific

expression of utrophin and promoter B drives expression in endothelial cells (Weir et al.,

2002). The core promoter A element contains recognition sites for the transcription factors

Ap2, Sp1 and Sp3, which orchestrate basal transcriptional activity (Perkins et al., 2001).

Importantly, the upstream promoter consists of motifs including the N-box, E-box and

NFAT site, which have all been shown to influence the expression of utrophin (Angus et

al., 2005; Gramolini et al., 1999; Perkins et al., 2001). The N-box localises the expression

of utrophin to the NMJ (Dennis et al., 1996). More recently, a PPRE element has been

described which is known to be important in regulating the transcription of genes involved

in lipid metabolism, oxidative respiration and promoting the formation of slower, more

oxidative muscle fibers (Miura et al., 2009).

A range of strategies have been used to upregulate utrophin. These include the use

of heregulin, which acts via the N-box motif of the utrophin A promoter (Krag et al., 2004)

and L-arginine, which results in an increase in utrophin expression as a result of increased

production of nNOS (Voisin & de la Porte, 2005). Jazz is an artificially generated

transcription factor designed to target a specific region in the utrophin A promoter (Corbi et

al., 2000; Di Certo et al., 2010), administration of a PPAR/

agonist GW501516 results

in a fast-to-slow fibre-type switch and concomitant increase in utrophin expression in mdx

mice (Miura et al., 2009). Biglycan is an extracellular matrix protein that most likely

increases utrophin expression by recruiting the protein to the sarcolemma without having

an effect on transcript levels (Amenta et al., 2011). Overexpression of CT-GalNac

transferase in mdxmice skeletal muscle results in increased carbohydrate glycosylation at

the sarcolemma and a resultant increase in extrasynaptic utrophin expression (Durko et al.,

2010). TAT-utrophin, a recombinant utrophin protein modified with the HIV-derived TAT



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protein transduction domain, improves delivery across the cell membrane (Sonnemann et

al., 2009). Overexpression of RhoA, a small GTPase, results in an increase in utrophin

expression with no change in transcription levels (Gauthier-Rouviere & Bonet-Kerrache,

2009). Although all these experiments have yielded positive results, none have been able to

achieve complete amelioration of the mdxdystrophic phenotype and the 3-4 fold

upregulation of utrophin thought to be necessary for complete correction.

Our laboratory has used the approach of finding drugs which increase utrophin

levels by exploiting a stable H2K mdxmyoblast cell line (Morgan et al., 1994) which

encodes 8.4 kb of the mouse utrophin promoter A lying 5' of the transcription start site and

linked to a luciferase reporter gene (Davies, Russell and Davies unpublished; Khurana &

Davies, 2003; Tinsleyet al., 2011). An initial screen of 5000 small compounds, carefully

designed to cover a broad range of biological space and omiting any structures containing

reactive or cytotoxic functionalities, was able to identify candidates which upregulated

utrophin gene expression in a dose-dependent manner. A secondary assay was used to

confirm that active compounds increased utrophin mRNA (and were not acting through

stablisation of the luciferase reporter). A final in vitroassay using human DMD patient

cell lines then confirmed increased utrophin protein expression. The in vivoeffect of

positive compounds was then assessed following intraperitoneal administration or oral

gavage in the mdxmouse. This work was done in partnership with Summit plc, who used

the cell line to find drugs which increased utrophin levels 5-fold. The chemistry was

optimized and resulted in one drug, SMT C1100, which showed therapeutic potential as

assessed by its prevention of pathology in the mdxmouse (Tinsley et al., 2011).

SMT C1100 targets the primary cause of the disease by reducing the level of

muscle membrane damage, as demonstrated by a reduction in force drop following

eccentric contractions (Tinsleyet al., 2011). Serum creatine kinase, muscle fibrosis and

necrosis are also reduced indicating that SMT C1100 diminishes the catastrophic secondary

pathology associated with the disease. Even when efficacy was tested in the more severe

exercised mdxmouse model, SMT C1100 treatment afforded increased muscle strength, a

partial reduction in the resistance to fatigue, and a significant amelioration of calcium-

dependent functional parameters (Tinsleyet al., 2011). Interestingly, the drug also



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demonstrated a synergistic effect when administered with prednisolone (Tinsleyet al.,

2011) the current gold standard in treatment of DMD patients in the clinic. SMT C1100

was taken into Phase I trials by BioMarin pharmaceuticals (as BMN-195). Although there

were no safety issues, the plasma levels of the drug were not high enough for the trials to

continue into patients. New formulations of the drug are currently being explored by

Summit plc with a view to taking this compound back into the clinic.

SMT C1100 demonstrates the principle of finding drugs which upregulate utrophin

and more screens are ongoing. Future screens will be based on a cell line which has the

luciferase gene knocked into exon 7 of the endogenous gene (Fairclough and Davies,

unpublished). This will enable screening of the promoter in its genomic context and

therefore should include most, if not all, of the regulatory elements, therefore better

mimicking the in vivosituation.

Concluding remarks

It is more than 20 years since the identification of the dystrophin gene and the development

of therapy has taken a long time. In spite of the challenges that remain, the current clinical

trials are looking very promising. Even if one strategy does not work efficiently enough, it

may be possible to combine these approaches. The next phase of DMD research looks to

be a very exciting one for scientists, clinicians and patients.

Acknowledgements

We are grateful to the Medical Research Council, Muscular Dystrophy Campaign,

Muscular Dystrophy Association USA, Association Francaise contre les Myopathies and

the Clarendon fund for their support of our work. We would also like to thank Aurelie

Goyenvalle for her comments on the manuscript and helpful suggestions towards the

figures.



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Figure 1



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49 51 5248

AO

Exon 51 skipping

49 5248

Shorter functional mRNA

TGA

49 51 5248

Exon 50 deletion in DMD mRNA with frameshift+PTC

4948

TGA

49 50 51 5248

Wild type dystrophin gene

49 50 51 5248

Full length functional mRNA

TGA

49 51 5248

Exon 51 nonsense mutation in DMD

49 5048

mRNA with PTC

50

49 51 5248

Read through of Exon 51

TGA

50

Compoundsuppressing nonsense

codons

TGA

49 50 51 5248

Full length functional mRNA

Figure 2

51 52

TGA

TGA

51 52

A

B

C

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