LILITE CONCEIÇÃO DA SILVA BARBOSA

DUCHENNE AND BECKER’S DYSTROPHINOPATHIES

A REVIEW OF THE LATEST THERAPEUTIC STRATEGIES AND REHABILITATION

PROGRAMMES

2010

LILITE CONCEIÇÃO DA SILVA BARBOSA

DUCHENNE AND BECKER’S DYSTROPHINOPATHIES

A review of the latest therapeutic strategies and rehabilitation

programmes

Porto, 2010

LILITE CONCEIÇÃO DA SILVA BARBOSA

DUCHENNE AND BECKER’S DYSTROPHINOPATHIES

A review of the latest therapeutic strategies and rehabilitation

programmes

Tutor: MANUEL JORGE ROCHA MELO PIRES

PhD (University of London)

Porto, 2010

Dissertation presented to the

Course of Integrated Masters in

Medicine to acquire the Master

Degree in Medicine.

LILITE CONCEIÇÃO DA SILVA BARBOSA

DUCHENNE AND BECKER’S DYSTROPHINOPATHIES

A review of the latest therapeutic strategies and

rehabilitation programmes

Approval date: __________________________________

Jury:

Prof. Dr. . ...................................................................

Prof. Dr. . ...................................................................

Prof. Dr. . ...................................................................

iv

SUMMARY

Duchenne and Becker muscular dystrophies are X-linked progressive

dystrophinopathies that result of genetic defects in dystrophin, the major

structural protein of the muscle. Their clinical patterns are similar and include

gradual generalized weakness and wasting of all voluntary muscles as well as

several other physical symptoms. Duchenne dystrophy differs from Becker

muscular dystrophy which has a later age of onset and a slower rate of

progression.

The correct diagnosis is very important to avoid misclassifications and it

relies both in clinical signs and in important tools such as measurement of

creatine phosphokinase, electromyography, muscle biopsy and genetic testing.

Therapeutic strategies for dystrophinopathies can be categorized into

three groups: molecular, cellular and pharmacological therapies. Molecular

therapy involves viral and non-viral approaches while cellular therapy involves

myoblast and stem cell strategies. Currently, pharmacological therapies are

supportive and play the main role in stopping disease‟s progression. They

involve a multidisciplinary approach early in the disease with the use of drugs/

molecules to improve the phenotype as well as rehabilitation programs including

physiotherapy or surgery.

Key Words: Dystrophinopathies; Molecular Therapy; Cellular therapy;

Pharmacological Therapy.

v

TABLE OF CONTENTS

List of Figures .......................................................................................vii

Introduction...........................................................................................8

Pathophysiology ...................................................................................8

Clinical Features ...................................................................................10

Diagnosis..............................................................................................11

Differential Diagnosis ...........................................................................13

Evaluation of Disease‟s Progression ....................................................14

Treatment .............................................................................................15

Molecular Therapy ................................................................................15

1. Viral-mediated Gene Therapy ................................................15

A. Adenovirus Vectors .....................................................15

B. Helper-dependent/gutted Adenoviral Vectors .............15

C. Adeno-associated Virus Vectors .................................16

D. Lentivirus Vectors .......................................................16

E. Homologous Recombination .......................................16

F. Utrophin Genetic Up-regulation ...................................16

2. Non-viral Gene Therapy .........................................................17

A. Plasmid DNA ..............................................................17

B. Antisense Oligonucleotides .........................................18

C. Chimeric RNA/DNA Oligonucleotides .........................19

Cellular Therapy ...................................................................................20

1. Myoblast Transplantation .......................................................20

2. Stem Cell Therapy .................................................................21

Pharmacological Therapy .....................................................................22

vi

1. Anti-inflammatory Drugs ........................................................22

A. Steroids .......................................................................22

B. Other Anti-inflammatory drugs ....................................25

2. Specific Anti-cytokine drugs ...................................................26

3. Anabolic Processes Stimulation .............................................26

A. Myostatin Regulation ..................................................26

B. Insulin-like Growth Factor I .........................................26

C. β2-agonists .................................................................27

4. Antibiotics ..............................................................................27

5. Anti-proteases ........................................................................28

6. Anti-oxidants ..........................................................................28

7. Pharmacological utrophin up-regulation .................................29

8. Supplements ..........................................................................30

9. Management of Complications ..............................................31

A. Respiratory .................................................................31

B. Cardiac .......................................................................31

C. Orthopedic ..................................................................32

i. Ambulant Children .............................................32

ii. Non-ambulant Children .....................................32

iii. Management of Scoliosis .................................32

Conclusion............................................................................................33

References ...........................................................................................34

vii

LIST OF FIGURES

Figure 1: Gower‟s Sign ........................................................................10

Figure 2: Muscle Biopsy (H&E staining) ...............................................11

Figure 3: Western Blot of Dystrophin ...................................................12

Figure 4: Dystrophin Staining ...............................................................13

8

INTRODUCTION

The muscular dystrophies are a group of inherited myogenic disorders

that share a set of clinical and pathological characteristics but differ in severity,

inheritance pattern, and molecular defect.

The most well known and prevalent of these disorders is Duchenne

Muscular Dystrophy (DMD), followed by Becker Muscular Dystrophy (BMD), a

clinically similar disorder but less severe. Both of them are X-linked progressive

Muscular Dystrophies with an incidence of 1/3500 male births in DMD and

1/30000 males in BMD “Emery (1991)”.

Duchenne Muscular Dystrophy was originally describe by Edward

Meyron in 1851 “Meyron (1851)” but it was only when Duchenne detailed the

clinical and muscle histology in 1861 that it became a recognized independent

disorder “Duchenne (1861)”. Almost 100 years later, in 1953 Becker described

an allelic variant to DMD with later age of onset and slower progression;

nowadays known as Becker Muscular Dystrophy “Becker (1953)”.

PPPAAATTTHHHOOOPPPHHHYYYSSSIIIOOOLLLOOOGGGYYY

DMD and BMD result of genetic defects in the major muscle cell

structural protein known as dystrophin, being also called Dystrophinopathies.

The dystrophin gene is the largest described to date, it‟s located on Xp21 and it

occupies almost 2% of that chromosome. Dystrophin is a physiological 427 kDa

protein located in the subsarcolemmal region of the muscle fiber and it‟s

responsible for stabilizing the plasma membrane against mechanical stress

during muscle fiber contraction “Hoffman et al. (1987)”. It is organized into four

structural domains: the N-terminal actin-binding, the central rod domain, the

cysteine-rich domain binding, and the carboxy-terminal “Hoffman et al. (1987)”

”Koenig et al. (1988)”. Dystrophin is expressed mainly in skeletal muscle but it

also can be found in smooth muscle cells, cardiac myocytes and, in lower

levels, in brain.

Nonstructural roles have been described for dystrophin, making it a

multifunctional protein. In one hand, the dystrophin complex serves as a

signaling scaffold that is responsive to extracellular stressors and several

9

signaling molecules interact with it “Brenman et al. (1995); Yang et al. (1995);

Abramovici et al. (2003); Oak et al. (2003)”. In the other hand, dystrophin is

thought to play a role in calcium homeostasis, resulting in the aberrant

hyperactivation of signaling cascades involved in the inflammatory response.

This apparent sensitivity of dystrophic muscle in triggering inflammation due to

aberrant calcium homeostasis may be detrimental to muscle cell survival and to

the potential introduction of therapeutics “Turner et al. (1991); Iwata et al.

(2003); Kumar et al. (2004)”.

Mutations in dystrophin gene comprise deletions (60-70% of cases),

duplications (10-15%) and point mutations (10-15%). In one third of the cases,

mutations occur de novo “Neri et al. (2007)”.

The gene-coded defects of dystrophin underlie DMD, which is marked by

absence or severe reduction of the protein, and BMD with presence of

dystrophin but defective one “Reitter and Goebel (1996)”. The threshold level of

dystrophin mRNA and protein sufficient to maintain proper membrane

organization and prevent muscle degeneration in humans is between 29% and

57%, if dystrophin is uniformly present in all muscle fibers “Neri et al. (2007)”.

This lower level is similar to the one found in previous transgenic experiences

on mdx mice “Wells et al. (1995)”. There is also a correlation between the

amount of dystrophin expressed and the clinical severity: absence or less than

3% of normal dystrophin results in severe DMD; from 3-10% correlates with

severe BMD; and from 20-30% of normal or altered dystrophin results mild BMD

“Hoffman et al (1988)”.

Only one highly homologous dystrophin protein (80%), utrophin or

dystrophin-related protein was described until now and it‟s encoded by a gene

in chromosome 6 “Khurana et al. (1990)”. In developing skeletal muscle,

utrophin showed to identify the entire sarcolemmal membrane. It was reported a

phenomenon of utrophin up-regulation in dystrophic muscle but this increase in

utrophin levels doesn‟t prevent muscle degeneration; it only delays the

progression of the disease “Vainzof et al (1995)”.

10

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In DMD, the onset is early in childhood, at 2-6 years of life, with a

waddling gait secondary to hip girdle muscle weakness. Very often parents note

that the child pushes on his knees in order to stand, sign known as Gower‟s

sign (Fig.1) “Gowers (1988)”. Cognitive impairment is usual and about 20% of

affected children have an IQ lower than 70. Calf enlargement (calf

pseudohypertrophy) is common, resulting of fatty and fibrotic infiltration of

degenerative calf muscles. Progressive weakness is seen in the proximal

musculature, initially in the lower extremities but later on also in the upper ones.

Around the age of 8 respiratory muscle‟s strength and ambulation capacity start

to decline, most of the patients being wheelchair bounded by the age of 10.

Scoliosis appears once wheelchair dependency is achieved which further

compromises pulmonary, cardiac and gastrointestinal function “Emery (1993)”.

In the past, patients developed terminal respiratory or cardiac failure due to

wheelchair bound and profound weakness by the early twenties; nowadays the

survival increased to late twenties “Simons et al. (1998)”.

BMD is characterized by a later age of onset, normally around 12 years;

distribution of muscle wasting and weakness similar to DMD but with a slower

rate of progression. The loss of ambulation may vary but mostly it occurs from

adolescence on, being the expectancy of life in BMD about 40 years of age with

90% of the patients alive beyond twenty years “Becker and Kiener (1955)”.

Fig.1: Gower‟s sign

11

DDDIIIAAAGGGNNNOOOSSSIIISSS

Measurement of serum creatine phosphokinase (CPK) is an easy and

sensitive test when suspecting of any muscle disease and correlates with the

amount of muscle degeneration. Patients with DMD and BMD have increased

levels of CPK since birth and they are 50 to 100 times higher than the reference

range. The neonatal screening of CPK for boys with family history and the

standard evaluation of CPK serum levels in boys with developmental

impairment are recommended since they decrease substantially the diagnosis‟

delay “Ciafaloni et al. (2009)”.

Electromyography, even though not diagnostic, is important to exclude

neurogenic causes of weakness.

Muscle biopsy, along with immunohistochemistry and Western Blot

analysis, is an essential adjunctive tool because in the majority of cases it‟s the

deciding factor for diagnosis before genetics studies. Muscle biopsy shows

increased variability of fiber size, rounding of fibers, fiber splitting, increased

numbers of central nuclei, hyaline fibers, muscle fiber necrosis, phagocytosis

and regeneration, and replacement by fat and fibrous tissue “Hilton-Jones

(2001)” (Fig.2).

a

q

b

q Fig.2: H&E (a) Normal muscle biopsy. (b) DMD fibers are smaller and

rounded. The fiber size is variable with many hypercontracted fibers. The

endomysial connective tissue is increased (fibrosis).

12

Western blot analysis for dystrophin in muscle allows the determination

of both the quantity and size of the molecule. In DMD dystrophin is absent or

levels less than 3% can be found. In 80% of patients with BMD, the molecular

weight of dystrophin is reduced to 20-90% of normal, 15% have reduced

quantity and 5% show an abnormally large protein “Hoffman et al (1988)”

(Fig.3). Muscle biopsy has the disadvantage of being an invasive procedure.

Fig.3: Western blot of dystrophin.

Lane 1: BMD; Dystrophin has reduced abundance but normal size.

Lane 2: BMD; Dystrophin has reduced size and abundance.

Lane 3: Normal; Dystrophin has normal size and amount.

Lane 4: DMD; Almost no protein is present.

Lane 5: DMD; Dystrophin has severely reduced abundance.

Immunostaining for dystrophin and utrophin on skin biopsies is also a

reliable tool for the diagnosis of dystrophinopathies “Tanveer et al. (2009)”.

Diagnosis can be achieved by genetic testing on a peripheral blood

sample. About 96% of mutations are identified by direct sequencing of the

dystrophin gene. The three main techniques are used in a 3-time approach:

first polymerase chain reaction (PCR) amplification to detect large deletions;

secondly detection of virtually all mutations (DOVAM) for point mutations; thirdly

multiplex amplifiable probe hybridization (MAPH) to define duplications. Since

the dystrophin gene is very large, most testing protocols only screen a part of

the gene. Thereby, a negative genetic test doesn‟t exclude the diagnosis and a

muscle biopsy is required “Dent et al. (2005)”.

Genetic counseling is very important and the first line test to determine

the female carrier status is a sensitive PCR for the same deletion/ duplication

observed in the affected child. With the possibility of germline mosaicism a

negative carrier test doesn‟t exclude the risk of mutation. Moreover, one third of

the cases represent new mutations.

For the prenatal diagnosis, multiplex PCR can detect around 93% of

deletions being simple, reliable and inexpensive. Multiplex ligation-dependent

probe amplification (MLPA) is important for detecting deletions in non-hot

regions/exons and duplications “Clemens et al. (1991); Li et al. (2009)”.

13

DDDIIIFFFFFFEEERRREEENNNTTTIIIAAALLL DDDIIIAAAGGGNNNOOOSSSIIISSS

There can be clinical and histopathologic overlap between muscular

dystrophies and inflammatory myopathies. Pathologic findings of inflammation

and major histocompatibility complex up-regulation, typical of inflammatory

myopathies, occur in some muscular dystrophies. Dystrophin levels should be

access by immunocytochemical staining in all cases “Nirmalananthana et al.

(2004)” (Fig.4).

Fig.4: Dystrophin staining. (a) Normal dystrophin staining around the rim of muscle fibers.

(b) Absent dystrophin in DMD. (c) DMD with one “revertant” fiber with dystrophin staining.

Limb-girdle muscular dystrophies are clinically similar to DMD, but occur

in both sexes due to autosomal recessive and autosomal dominant inheritance.

Testing for deficiency of proteins from the transmembrane sarcoglycans

complex is indicated in dystrophin-positive dystrophies (Erazo-Torricelli (2004)”.

Emery-Dreifuss muscular dystrophy is associated with limb contractures

and cardiac arrhythmia. X-linked recessive forms are identified by emerin deficit

on immunohistochemistry, autosomal dominant and autosomal recessive forms

are both exclusively identified by DNA study “Erazo-Torricelli (2004)”.

Spinal Muscle Atrophy is characterized by poor muscle tone and

symmetric muscle weakness sparing the face and ocular muscles due to

anterior horn cell loss. The definitive diagnosis relies on DNA analysis of

mutations in the Survival of Motor Neuron 1 gene (SMN1) “Monani (2005)”.

a

q

c

q

b

q

14

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Annual neurological, respiratory and cardiological assessments should

be co-ordinated via a centralized rehabilitation unit.

From the time of diagnosis boys must be assessed once to twice a year

by therapists with special experience in neuromuscular disorders. The interval

between assessments depends on boy‟s age, disease‟s progression and his

functional ability “Bushby et al. (2005)”.

Respiratory surveillance should include: serial spirometry accessing

forced vital capacity (FVC) and peak expiratory flow (PEF) to document the

progression of respiratory muscle weakness; serial measurement of nocturnal

oxycapnography/polisomnography, once clinical signs of nocturnal

hypoventilation; serial measurement of peak cough flow; chest and spine

radiograph for monitoring scoliosis‟ degree and chest deformities “Finder et al.

(2004)”.

Cardiac investigation (echocardiogram and electrocardiogram) is

indicated at diagnosis, every 2 years thereafter to age of 10 and then annually,

or more often, if abnormalities are detected “American Academy of Pediatrics

(2005)”.

15

TTRREEAATTMMEENNTT

Therapeutic strategies for dystrophinopathies are divided into 3 groups

based on their approach: molecular, cellular and pharmacological therapy.

MMMOOOLLLEEECCCUUULLLAAARRR TTTHHHEEERRRAAAPPPYYY

1. VIRAL-MEDIATED GENE THERAPY

The aim is to deliver DNA encoding dystrophin or other therapeutic

genes (utrophin) to muscle. To date most dystrophin-delivery approaches are

hampered by the gene‟s large size (it‟s larger than the cloning capacity of most

current viral vectors), immune responses to the protein and viral antigens, and

difficulty finding an effective delivery system. Important advances were made in

identifying key regions of the gene in truncated dystrophin gene construction,

optimal vectors for gene delivery and better delivery methods.

A. Adenovirus vectors

The first gene transfer vectors derived from adenovirus and, despite their

success in delivering dystrophin up to 50% to myofibers, they were too small to

fit the entire coding sequence of dystrophin. Functional studies of the gene in

mdx mice showed that multiple regions of dystrophin can be deleted in various

combinations to generate highly functional mini- and microdystrophin genes

with the advantage of being within viral/plasmid cloning capacities “Fabb et al.

(2002)”. One important problem was still remaining; the strong immune

response they caused “Smith (1995)”.

B. Helper-dependent/gutted adenoviral vectors

To overcome some of the limitations with adenovirus vectors, helper-

dependent or gutted adenoviral vectors, which are deleted for all of the viral

protein coding sequences, started to be used. Although a weaker immune

reaction was induced, they had to cross the basal lamina of muscle fibers

reducing the efficiency of transduction. A co-deliverance of immunomodulatory

molecules such as CTL4AIg was suggested to face some of these problems

“Parks et al. (1996); Dudley et al. (2004); Jiang et al. (2004)”.

16

C. Adeno-associated virus vectors (AAV)

Another promising viral vector is AAV given its nonpathogenicity, broad

tropism and infectivity and long-term persistence. These vectors appear to be

more efficient for transducing adult fibers, especially if delivered systemically

together with factors that increase vascular permeability. They can only

accommodate minidystrophin which gives good functional rescue when

replacing dystrophin in transgenic mdx mice “Wang et al. (2000)”.

D. Lentivirus vectors

Additional viral vectors being tested for gene transfer to muscle are the

HIV-derived lentiviral vectors. They have been found to stably transduce post-

mitotic cells with expression lasting up to 2 months with no associated immune

response “Odom et al. (2007)”.

E. Homologous Recombination (HR)

Other approach is called gene targeting, which means the replacement of

an endogenous DNA segment with a homologous, exogenous segment of DNA

by HR. A major advantage of the HR gene targeting approach is that the risk of

random integration as is presented with retroviral mediated gene replacement

can be avoided or minimized; this has been accomplished using helper-

dependent Adenovirus as a vector platform “Odom et al. (2007)”.

F. Utrophin genetic up-regulation

In skeletal muscle, utrophin is detectable in early fetal development over

the sarcolemma where during development it is gradually replaced by

dystrophin. This different expression proposed that utrophin may be the

embryonic/neonatal form of dystrophin thus; utrophin up-regulation would allow

replacement of dysfunctional or absent dystrophin. Furthermore, this approach

benefits from the lack of neoantigen production and subsequent immune

response that might result with the delivery of dystrophin “Miura and Jasmin

(2006)”.

17

2. NON-VIRAL GENE THERAPY

Three different methodologies have been pursued: 1) plasmid DNA to

deliver dystrophin cDNA constructs; 2) antisense oligonucleotides (AONs) to

induce exon skipping of the dystrophin pre-mRNA; 3) RNA/DNA chimeric

oligonucleotides for genome editing and gene correction. The main

disadvantage of non-viral vectors is the delivery mechanism but they also have

the potential to induce immune responses, although in a lower level. It is

considered that non-viral vectors will represent a less expensive and safer

mode of therapy “Rando (2007)”.

A. Plasmid DNA

This therapy is based on the observation that naked plasmid DNA,

delivered to skeletal muscle by direct injection, is taken up in the myofiber

where plasmid genes are expressed. Intramuscular or intravenous/arterial

injection of a therapeutic plasmid into mdx mice result in dystrophin expression

in up to 10% of the muscle fibers “Acsadi et al. (1991); Liang et al. (2004)”.

There are several positive aspects of this therapy including already

existing scaled-up production processes, applicability to DMD patients

regardless of mutation, and simplicity. Still, controversial issues exist and the

expected duration of plasmid gene expression is one of them; in human

treatment this expression would be necessary to last for years/decades.

Research leading to plasmid persistence is being made by introducing the

dystrophin plasmid into a single site in the genome with the use of integrases.

Ideally the integrases should be site-specific but currently they aren‟t which may

lead to insertional mutagenesis. Another concern regards the distribution of the

vector and thus the therapeutic protein along the length of muscle fibers in a

targeted muscle. Any fiber not expressing dystrophin at therapeutic levels along

its length will eventually succumb; thus the importance of quantifying dystrophin

expression not only in the muscle cross-sections but also along the longitudinal

axis of the muscle “Molnar et al. (2004); Bertoni et al. (2006)”.

18

B. Antisense oligonucleotides (AONs)

The possible therapeutic benefit of dystrophin transcript splicing

modifications is based on the fact that most cases of DMD are caused by

deletions in the dystrophin gene that lead to frame-shift mutations in the

transcript. This led to the idea of using AONs to alter splicing so that the open

reading frame is restored and the severe DMD phenotype is converted into a

milder BMD phenotype. Recent studies have clearly established the potential of

AONs induced exon-skipping approach since dystrophin expression was

enhanced with repeated injections and no immune response was elicited

“McClorey et al. (2006)”. It has been estimated that 70% of patients with DMD

caused by intragenic deletions could benefit from this therapy. Phase I clinical

trials with AONs in humans are being conducted to test intramuscular injections

of AONs for safety and efficacy “Muntoni et al. (2005)”. Recently, a study

reported that the application of multiexon skipping may provide a more uniform

methodology for a larger group of patients with DMD “Aartsma et al. (2004)”.

This approach corrects all dystrophin isoforms and maintains the original

tissue-specific gene regulation however, its application requires an accurate

molecular diagnosis, a rational plan for skipping one or more exons to produce

an in-frame transcript, evidence that targeting AONs to specific sequences will

result in the desired transcript, and finally the design of AONs vectors

specifically for that particular mutation “Wilton and Fletcher (2008)”. It also has a

transient therapeutic effect and AONs therapy would require multiple treatments

each year. One solution was combining AONs technology with viral expression

approaches to achieve sustained expression of antisense vectors and long-term

exon skipping but this entails the challenges of viral-mediated gene therapy

“Goyenvalle et al. (2004)”.

19

C. Chimeric RNA/DNA oligonucleotides

Chimeric RNA/DNA oligonucleotides can be used to direct the correction

of a mutation by inducing preferential gene conversion from a mutant to a

functional allele. Such genome editing potential could be applied to the

correction of point mutations responsible for dystrophin deficiency in

approximately 15% of patients with DMD. Injection of chimeric oligonucleotides

in mdx mice showed correction at the genomic and transcript levels, and

restoration of dystrophin protein expression was observed both in vivo and in

vitro “Rando et al. (2000); Bertoni and Rando (2002)”. To benefit more patients

with dystrophin defects other than point mutations, broader applications of this

approach have been envisioned. Recently, attempts to correct mutations that

disrupt the translational reading frame were made by targeting the exon/intron

boundaries of the mutated exon 23 in mdx mice. It resulted in skipping/removal

of this exon and production of a truncated but functional protein. The use of this

technique has to overcome similar problems described in the AONs therapy,

such as the location of the particular splice site to be targeted “Bertoni et al.

(2003)”. The major challenge concerning chimeric compounds is the low

efficiency observed of this technology with current vectors “Rando et al. (2000);

Bertoni et al. (2005)”. Current research is focusing on the enhancement of the

molecular mechanisms that mediate the gene editing, including modifications of

the oligonucleotides as well as enzyme systems and cellular processes involved

in the repair mechanism “Bertoni et al. (2006).”

Perhaps the greatest advantage is that it results in a permanent

correction of the genetic defect in myonuclei allowing sustained therapeutic

benefit as long as the targeted myonuclei persist.

20

CCCEEELLLLLLUUULLLAAARRR TTTHHHEEERRRAAAPPPYYY

Cell-based strategies comprise two main subjects of investigation:

transplantation of myoblasts from a healthy donor expressing normal

dystrophin; use of stem cells (ST) obtained from the patient that must be

„genetically corrected‟ in vitro. Recent works suggest the most promising

possibility for the treatment of DMD being a combination of different

approaches, such as gene and stem cell therapy “Farini et al. (2009)”.

1. MYOBLAST TRANSPLANTATION

This procedure involves injecting or transplanting donor muscle precursor

cells (myoblasts) into a dystrophic host. The goal is to induce expression of

dystrophin through fusion of dystrophin-positive myoblasts of the donor with

dystrophin-deficient muscle fibers of the host. Although shown to be promising

in mdx mice “Ikezawa et al.(2003)”, human trials didn‟t demonstrate objective

benefits with low levels of dystrophin expression “Mendell et al. (1995);

Partridge (2002)”. Myoblast transplantation has several limitations, including

immune rejection, poor cellular survival rates (80% undergo a rapid and

massive death soon after the injection) and lack of dispersion of injected cells

which limits the effect to the injection area “Gussoni et al. (1997); Mouly et al.

(2005); Skuk et al. (2006)”.

A recent study concluded that fetal myogenic cells can be successfully

isolated and expanded in vitro from human fetal muscle biopsies. Cells from a

16-week-year old fetus showed higher growth capacities when compared to

cells from 13 and 30 year old donors. This form of treatment is controversial due

to ethical issues regarding the use of fetal tissue “Hirt-Burri et al. (2008)”.

21

2. STEM CELL THERAPY

Different types of stem cells have been explored for the treatment of

DMD and the ideal scenario of a bone-marrow derived stem cell that circulates

to reach all muscles has proved clinically irrelevant to date “White and Grounds

(2003)”. Satellite cells represent the oldest known adult stem cell niche; they

reside beneath the basal lamina of skeletal muscle fibers and their two critical

properties are self-renewal and capacity to turn into various lineages of cells.

Satellite cells exhibit substantial phenotypic and functional heterogeneity,

evident through differences in their cell-surface marker expression, induction of

myogenic transcription factors, and in vivo and in vitro proliferation

characteristics “Rouger et al. (2004)”.

In late 1990s, were described cells likely to be more primitive than

satellite cells within muscle, nowadays called side population (SP) “Montanaro

et al. (2004)”. These cells demonstrated potential plasticity to myogenic and

hematopoietic lineages suggesting that to undergo myogenic commitment they

require the environment provided by myogenic cells “Asakura et al. (2002);

Camargo et al. (2003)”. They can be systemically delivered, intravenous or

intra-arterially, and then they extravasate from vessels into skeletal muscle,

engraft into muscle tissue and deliver their genetic material to host myofibers.

The mechanisms by which SP can extravasate from vessels and engraft in

skeletal muscle were studied and enhancement of progenitor cell engraftment

was achieved with intra-arterial SP transplantation “Perez et al. (2009)”.

Blood vessels were identified as a promising source of novel stem cells

called mesangioblasts. They can be delivered through blood and have a striking

capacity to form muscle and repopulate diseased skeletal muscles.

Mesangioblast therapy was studied in mice and in dogs with health

improvement of the dystrophic animals “Galvez et al. (2006); Sampaolesi et al.

(2006)” but in human treatment controversy still exists “Davies and Grounds

(2006)”.

A promising recent study showed that it is possible to transduce SP cells

from a dystrophic mouse with a lentiviral vector expressing human

microdystrophin, and reconstitute a few dystrophic fibers with the human protein

following intra-vascular delivery “Bachrach et al. (2004)”.

22

PPPHHHAAARRRMMMAAACCCOOOLLLOOOGGGIIICCCAAALLL TTTHHHEEERRRAAAPPPYYY

Presently supportive therapies are the main tool in reducing morbidity,

increasing quality of life and prolonging lifespan. Pharmacological approaches

involve the use of drugs/ molecules to improve the disease phenotype by

targeting specific components of the pathophysiology. Since the targets are

specific to a pathological defect, a combination of approaches is required which

enhance the probability of drug interactions and adverse effects.

1. ANTI-INFLAMMATORY DRUGS

A. Steroids

The relevant steroids in neuromuscular practice, prednisone/

prednisolone and deflazacort, have a predominant glucocorticoid action. They

showed potential for providing temporary improvement in two outcome

measures: prolongation of ambulation and enhancement of muscle strength and

function. Steroids demonstrated to stabilize muscle strength and function from

six months up to two years. Long-term pulmonary and cardiac benefits were

also reported but still need to be confirmed “Manzur et al. (2008)”.

Corticosteroids have a catabolic effect on muscle (non-exercised muscle)

preserving existing muscle fibers and reducing inflammation though, their exact

mechanism of action in dystrophic skeletal muscle is unknown. Several

possibilities based mainly on observations in mouse models were proposed and

include: differential regulation of genes in muscle fibers “Muntoni et al. (2002)”;

decrease in the number of cytotoxic/suppressor T cells “Kissel et al. (1991)”;

reduction of cytosolic calcium concentrations “Vandebrouck et al. (1999)”;

increasing laminin expression and myogenic repair “Anderson et al. (2000)”;

retarding muscle apoptosis and cellular infiltration “Kojima et al. (1999)”;

inhibition of muscle proteolysis “Rifai et al. (1995)”; protecting against

mechanically induced fiber damage “Jacobs et al. (1996)”; enhancing

dystrophin expression “Hardiman et al. (1993)”; shift the fiber type towards the

fast-twitch fibers “Fisher et al. (2005)”; increasing muscle levels of taurine and

creatine “McIntosh et al. (1998)”.

The first randomized, double-blind placebo controlled study on the use of

corticosteroids in DMD was reported in 1989. It compared a 6-month trial of

23

daily prednisone at doses of 0.75mg/kg and 1.5mg/kg where the 0.75 mg/kg

daily dose had the most favorable profile “Mendell et al. (1989)”. Later on, the

same group compared doses of 0.3mg/kg/day and 0.75mg/kg/day to placebo

and the lower dose of 0.3 mg/kg daily was less beneficial “Griggs et al. (1991)”.

Daily administration versus alternate day dosing was also studied and it was

found that daily dosing at 0.75mg/kg maintained the effects longer than

2.5mg/kg on alternate days “Fenichel et al. (1991)”.

Deflazacort is an oxazolone derivative of prednisone and the therapeutic

equivalence is about 1.2 mg deflazacort to 1.0 mg prednisone. A study

compared two treatment deflazacort protocols, one using 0.6mg/kg/day for the

first 20 days of the month and the other 0.9 mg/kg/day, determining the more

effective dose being 0.9 mg/kg/day. This study confirmed the long-term benefits

of deflazacort on mobility in both treatment protocols compared to boys not

treated with deflazacort. At the present deflazacort is only available in some

countries “Biggar et al. (2004)”.

Several comparison studies of prednisone and deflazacort have been

performed and one of the latest shows similar benefits to both treatments.

Deflazacort was also much more expensive and many families chose the

prednisone regimen. Regarding to side-effects, excessive weight gain was more

common with prednisone and asymptomatic cataracts with deflazacort “Balaban

et al. (2005)”.

Controversies still exist regarding to starting age of corticosteroids,

clinical criteria to start it, which corticosteroid, which dose and which regimen,

and when to discontinue corticosteroids. Immunizations schedule is generally

thought to be a reason to hold off initiating corticosteroids until 4 years of age.

Another reason is that under 2 years of age children are still gaining motor skills

making steroids unrecommended. Usually clinicians should propose initiation of

steroids at age 4–8 years, when a plateau phase (there is no longer progress in

motor skills and prior to decline) can be identified. Steroids are still advised

once a decline in motor skills is achieved but the benefits may be significantly

reduced “Bushby et al. (2010)”.

Systemic side-effects of steroid therapy in dystrophinopathies include

those commonly seen with chronic steroid use. There have been reports of the

following adverse effects: weight gain, behavioral changes, cushingoide

24

appearance, excessive hair growth, acne, osteoporosis/fractures,

hyperglycemia/glycosuria, hypokalemia, hypertension, gastrointestinal side-

effects, increased appetite, cataracts, sepsis and death “Manzur et al. (2008)”.

Maintenance of a daily schedule is appropriate when the child‟s motor function

is stable or in decline and if any corticoid side-effects are manageable and

tolerable. A regimen in alternate days is an option if unmanageable side-effects

develop in a daily dose regimen and they don‟t ameliorate with dose reduction.

In case of intolerable side-effects despite the regimen, a dose reduction of

approximately 25–33% is suggested and a clinical reassessment in 1 month is

necessary to determine whether side-effects have been controlled or not. If the

adjustments to dose and/or schedule regimens prove ineffective in making any

significant side-effects sufficiently manageable, then it is necessary to

discontinue therapy despite the state of motor function. Management for weight

gain should be accompanied by dietary advice and if obesity is of concern (>10-

20% over estimated normal weight for height over a 12-month period) switching

treatment from prednisone to deflazacort should be considered. Behavioural

changes should be supported by psychological input and advice on bone health

should be provided alongside with monitoring of fracture frequency. Vertebral

fractures can be treated with intravenous bisphosphonates under the guidance

of an expert in bone metabolism however there isn‟t sufficient evidence to

recommend the use of prophylactic oral bisphosphonates. Prophylaxis relies on

advice about calcium and vitamin D intake through diet and sunshine with

supplements of vitamin D3, if 25-hydroxy vitamin D is <32 nmol/L, and calcium

to reach an intake of 1000 mg/day. Concomitant treatment with non-steroidal

anti-inflammatory agents should be avoided and the use of antacids is

recommended in the presence of gastrointestinal complaints. Annual

ophthalmological examination and blood pressure/glucose intolerance

surveillance at each clinic visit are also advised “Manzur et al. (2008)” “Bushby

et al. (2010)”.

25

B. Other anti-inflammatory drugs

Other immunosuppressive drugs demonstrated benefits in mdx mice

leading to increasing recognition for the damaging role of inflammation in DMD.

Clinical trials on dystrophic muscle with the use of cyclosporine proved

beneficial through direct actions on skeletal muscle by inhibiting calcineurin and

ameliorating impaired mitochondrial metabolism. This demonstrated that

cyclosporine treatment significantly normalized many functional, histological,

and biochemical endpoints by acting on events that are independent or

downstream of calcium homeostasis. Muscle strength increased with a minimal

dose of 5 mg/kg/day, however, cyclosporine exerts multiple dose-dependent

effects and a correct dosage must be established especially when administered

to young dystrophic patients during muscle development “Sharma et al. (1993);

De Luca et al. (2005)”.

Another potential anti-inflammatory drug that has attracted attention is

the phosphodiesterases inhibitor pentoxifylline. Pentoxifylline is claimed to have

an anti-inflammatory, anti-oxidant and anti-fibrotic action; it reduces Tumor

Necrosis Factor-alpha (TNF-α) production in vitro, reduces fibrosis and may

also play a role in normalizing blood flow in dystrophic muscle. It was also

shown that pentoxifylline counteracts the abnormal activity of calcium channels

responsible for high sarcolemmal calcium permeability of dystrophic myofibers,

suggesting a possible amelioration of dystrophic condition through alternative

pathways. Despite this apparent modulation of many different targets, it was

proposed a unique mechanism of action residing in pentoxifylline ability to

inhibit specifically several isoforms of phosphodiesterases in different tissues.

Until now no significant side-effects were reported with the use of pentoxifylline

but, considering the function of phosphodiesterase enzymes and the key role of

cyclic nucleotides for modulating vital functions, it‟s important to use it carefully

with special attention to dose and administration routine “Burdi et al. (2009)”.

Oxatomide and cromolyn are other anti-inflammatory drugs that block

mast cell degranulation. They have shown benefits in mdx mice, indicating that

mast cell products, including tumor necrosis factor-alpha (TNF-α), have

detrimental effects in dystrophic muscle. However, further studies must be

performed to corroborate this hypothesis “Granchelli et al. (2000); Radley and

Grounds (2006)”.

26

2. SPECIFIC ANTI-CYTOKINE DRUGS

A new approach involves the modulation of specific cytokines, which has

been very successful clinically in some severe inflammatory disorders. TNF-α is

a key pro-inflammatory cytokine that stimulates the inflammatory response and

its specific pharmacological blockade may reduce muscle necrosis. Satisfactory

results in dystrophic muscle were obtained with the use of two different

antibodies against TNF-α, infliximab and etanercept. In 2004, a study with

infliximab-treated mdx mice showed reduction in the onset, intensity of necrosis

and dystropathology “Grounds and Torrisi (2004)”. Later studies demonstrated

that etanercept reduced effectively plasma CPK with physiological benefits in

muscle strength and chloride channel function “Hodgetts et al. (2006); Pierno et

al. (2007)". This new strategy has the important benefit of few related side-

effects due to its target high specificity.

3. ANABOLIC PROCESSES STIMULATION

A. Myostatin regulation

Myostatin is a member of the Transforming Growth Factor-beta family

(TGF-β) and it‟s a potent, negative regulator of functional muscle mass.

Deletions of the myostatin gene cause muscle cell hypertrophy “Schuelke et al.

(2004)” and in the mdx mouse, blocking myostatin lead to an increase in body

weight as well as muscle mass, size and strength. Inhibition of the myostatin

gene by injecting blocking antibodies is predicted to improve the disease

phenotype in a variety of myopathies. In treated animals a significant reduction

in muscle fiber degeneration and serum CPK levels was observed but the exact

mechanism whereby myostatin improves muscle function is not yet known.

Studies showed that an increase in the expression of utrophin appears not to be

involved in the process “Bogdanovich et al. (2002)”.

B. Insulin-like growth factor I (IGF-I)

IGF-I is an example of a positive regulator of muscle growth. Although

the presence of IGF-I can‟t correct the primary defect in muscular dystrophy, it‟s

possible that it can play an important role defending against the secondary

symptoms of the disease. Several different studies of normal muscle showed

27

that IGF-I is effective in increasing muscle mass and strength, promoting muscle

regeneration, and preventing apoptosis. It was also described that muscle-

specific IGF-I expression is beneficial to dystrophic muscle by preventing

fibrosis in addition to promoting functional hypertrophy. The combined effect of

enhanced repair and decreased wasting might lead to greater functional

capacity over time, where there is a reduction in the proportion of maximal effort

needed to produce a required force making the muscle less likely to be

damaged by normal activity. As so, the increased rate of muscle regeneration

may be a therapeutic alternative to replace the missing or defective dystrophin

complex member in the dystrophinopathies “Barton et al. (2002)”.

C. β2-agonists

Beta-2 adrenergic agonists were shown to induce muscle hypertrophy

and prevent atrophy after physical and biochemical insults in animals. A clinical

trial in a boy with DMD showed that albuterol produced a modest increase in

muscle strength without significant side-effects “Fowler et al. (2004)”. Recent

studies reported that formoterol has more powerful anabolic effects on skeletal

muscle than the older generation of β2-agonists, but inconsistency still exists

regarding to adverse effects of this therapy “Harcourt et al. (2007)”.

4. ANTIBIOTICS

Approximately 15% of DMD cases and most BMD cases are due to the

formation of a premature stop codon within the coding sequence of dystrophin.

Gentamicin is an aminoglycoside antibiotic that interferes with the ability of the

ribosome to recognize a given stop codon allowing translation to continue past

(suppress) a mutation in a given gene. This seems to be the only current

pharmacological strategy aiming to correct the primary defect. Gentamicin-

treated mdx mice showed both an increase in dystrophin expression, up to 10–

20%, and functional improvement. Protection against contractile injury was also

reported as a result of sarcolemmal dystrophin localization “Barton-Davis et al.

(1999)”. Concerning to human gentamicin trials, although an initial decrease in

CPK levels was noted, there was no increase in dystrophin expression as in

animal‟s results “Wagner et al. (2001); Dunant et al. (2003)”.

28

5. ANTI-PROTEASES

Dystrophin deficiency results in elevated Ca2+ influx and impaired Ca2+

homeostasis, which in turn activates calpain proteases that may account for

myofiber degeneration. A pioneer study suggested a potential therapeutic effect

with a new compound, BN 82270 (Ipsen), which is a membrane-permeable

prodrug of a chimeric compound (BN 82204) dually acting as calpain inhibitor

and anti-oxidant. It demonstrated increase in muscle strength, reduction in

serum CK and fibrosis of the mdx mice diaphragm “Burdi et al. (2006)”.

However, recent data showed that calpain enzymes perform only limited

proteolysis of target substrates being unlikely the predominant contributor of

muscle protein breakdown. Despite this, calpain enzymes play an important role

initiating the breakdown of myofibrillar proteins by releasing protein fragments

that become suitable substrates for the ubiquitin-proteasome system. Two

classes of muscle-cell permeating dual calpain/proteasome inhibitors were

developed and studied. Tripeptidic calpain/proteasome inhibitors showed

superior proteasome inhibition when compared to dipeptidic inhibitors as well as

higher potency in inhibiting cellular Ca2+-induced proteolysis induced by Ca2+

influx. Given their inhibitory activity and lower cellular toxicity, these compounds

have a potential benefit in dystrophinopathies “Briguet et al. (2008)”.

6. ANTI-OXIDANTS

Several findings suggest that oxidative stress might be involved in the

dystrophic process. Free radical injury may contribute to loss of membrane

integrity in muscular dystrophies and dystrophic muscle cells have an increased

susceptibility to reactive oxygen intermediates “Rodriguez et al. (2003)”.

Coenzyme Q10 (CoQ10; also called ubiquinone) is a powerful

antioxidant and mitochondrial respiratory chain co-factor. Previous double blind

trials have shown some efficacy but further studies are needed. At the present a

phase III clinical trial is currently assessing the effect of CoQ10 (serum level

greater than 2.5 μg/ml) and prednisolone (0.75 mg/kg/day) in wheelchair-

dependent patients with DMD “http://www.clinicaltrials.gov/“.

Green tea polyphenols are also powerful antioxidants and along with its

active constituents may improve dystrophinopathies prognosis. In an

29

experiment varying concentrations of green tea extract were given to mice for

four weeks, beginning at birth. It was determined that the extract significantly

reduced the degradation of certain muscles and noted that higher doses

correlated with greater inhibition of decline. There was also biochemical

evidence that green tea extract reduced oxidative stress in muscle cells

“Dorchies et al. (2006)”.

It has also been suggested a role for nuclear factor (NF)-қB in muscle

degeneration. The effects of blocking NF-қB by inhibition of oxidative stress/lipid

peroxidation on the dystrophic process in mdx mice were studied using a strong

antioxidant and lipid peroxidation inhibitor, IRFI-042. It was shown that IRFI-042

reduced muscle necrosis and enhanced regeneration by blunting NF-қB DNA-

binding activity and TNF-α expression in the dystrophic muscles “Messina et al.

(2006)”.

7. PHARMACOLOGICAL UTROPHIN UP-REGULATION

The high degree of structural similarity between dystrophin and utrophin

led to the speculation that utrophin might be able to replace dystrophin in DMD

and reconstitute the dystrophin associated protein complex (DAPC) “Tinsley

and Davies (1993)”. Two promoters (A and B) have been described at the 5‟

end of the utrophin gene with utrophin A being the expressed one at the

neuromuscular junction; the expression of utrophin B is mainly confined to the

vascular endothelia “Weir et al. (2002)”.

The utrophin promoter A is transcriptionally regulated in part by

heregulin-mediated, extracellular signal-related kinase-dependent activation of

the GABPα/β transcription factor complex. Studies showed amelioration of

phenotype but not as dramatic as that reported by using transgenic means. Yet,

it has a fundamental advantage of obviating the immune and delivery problems

associated with germ-line modification and/or somatic gene therapy “Krag et al.

(2004)”.

Stimulation of Calcineurin is thought to increase extrasynaptic expression

of utrophin-A leading to restoration of DAPC and attenuation of the dystrophic

pathology “Chakkalakal et al. (2004)”.

30

Treatment of mdx mice with nitric oxide donors or substrates of nitric

oxide synthase (NOS) such as L-arginine increased the expression of utrophin

with concomitant morphological corrections of muscle. Neuronal nitric oxide

synthase (nNOS) catalyzes the production of nitric oxide and citrulline from the

substrate L-arginine. Studies show that the loss of nNOS is a major contributing

factor to many of the pathological changes in DMD “Voisin et al. (2005)”.

A recent work showed that the utrophin 5‟ end confers IRES (internal

ribosome entry sites)-mediated translational control during regeneration of

skeletal muscle fibers. Some viral and eukaryotic cellular mRNAs can be

translated via internal initiation making the stabilization of utrophin or the post-

transcriptional control of utrophin expression additional targets for therapeutic

intervention “Miura et al. (2005)”.

8. SUPPLEMENTS

Creatine is an “energy precursor” naturally produced by the body.

Transformed by the body into phosphocreatine, it enters muscle cells and

promotes protein synthesis while reducing protein breakdown. Data suggested

that supplemental creatine can improve muscle performance and strength,

decrease fatigue, and slightly improve bone mineral density. Glutamine is

involved in many metabolic processes and it is an important energy source for

many cells. A recent study showed inconclusive results but it couldn‟t exclude a

disease-modifying effect of creatine and glutamine in younger DMD “Escolar et

al. (2005)”.

Taurine is abundant in normal skeletal muscle and is believed to exert

both long- and short-term control over the functionality of ion channels.

Supplementation of taurine counteracts exercise-induced weakness after

chronic exercise and ameliorates macroscopic chloride conductance (an index

of degeneration-regeneration) in mdx mice muscles “De Luca et al. (2003)”.

A combination of creatine monohydrate, conjugated linoleic acid, alpha-

lipoic acid and betahydroxy-beta-methylbutyrate improved strength and

decreased fatigue in the mdx mice. This combination was better than any

individual supplement alone and the combination with prednisone provided the

best results “Payne et al. (2006)”.

31

9. MANAGEMENT OF COMPLICATIONS

A. Respiratory

Effective airway clearance is critical to prevent atelectasis and

pneumonia that can lead to respiratory failure and death. Cough peak flows

correlate directly to the ability to clear secretions from the respiratory tract and

the use of assisted cough technologies is recommended whether there‟s clinical

signs of ineffective clearance, the peak cough flow is less than 270 L/minute or

when maximal expiratory pressures are less than 60cm H2O. Mechanical

insufflation-exsufflation has shown to be more effective than manual

techniques.

Another major complication of dystrophinopathies is a sleep-disordered

breathing. The main recommendation is to use nasal continuous positive airway

pressure (CPAP) in patients with obstructive sleep apnea syndrome but without

nocturnal hypoventilation; and to use nasal intermittent positive pressure

ventilation (NIPPV) along with bilevel positive airway pressure (BiPAP)

generator or mechanical ventilator to treat sleep-disorder breathing and

nighttime hypoventilation. Avoidance of supplemental oxygen is crucial because

it can suppress respiratory drive and worsen the defect. Daytime ventilation

should be considered when measured PCO2 exceeds 50 mmHg or when

hemoglobin saturation remains < 92% while awake. Non-invasive techniques

like mouth-piece intermittent positive pressure ventilation are preferred but

tracheostomy remains an option when others are contraindicated “Finder et al.

(2004)”.

B. Cardiac

Dilated Cardiomyopathy (DCM) occurs in up to 90% of boys with DMD

aged ≥18 years. It becomes symptomatic only in a minority and it‟s the cause of

death in 20% of the cases. Recently, positive results were found with the use of

angiotensin converting enzyme inhibitors, mainly perindopril, and/or beta-

blockers in patients with early cardiomyopathy. However optimal timing for

introducing therapy for DCM remains undetermined “Bouhouch et al. (2008)”.

32

C. Orthopedic

The main goal in the treatment of DMD is to maintain ambulation for as

long as possible and to anticipate and manage the associated complications,

such as joint contractures and scoliosis. There are several physical

interventions recommended according to the stage of the disease.

i. Ambulant children: active, active-assisted or passive daily

stretching at the ankle, knee and hip are necessary to prevent contractures.

Moderate levels of active exercise, particularly hydrotherapy or swimming, are

recommended. Night ankle-foot-orthoses (AFOs) should be provided at the loss

of ankle dorsiflexion, when there is loss of normal grade of dorsiflexion and

before the foot can only achieve plantagrade. Daytime AFOs are not suggested

for ambulant children as they compromise their ability to walk. KAFOs (knee-

ankle-foot-orthoses) can be considered to delay contracture development and

prolong ambulation in the late ambulatory stage. Surgical lengthening of early

contractures is also an option and it has been performed in children as young

as 4-7 years. Surgical intervention is thought to prolong ambulation by 1 to 3

years “Eagle (2002); Bushby et al. (2010)”.

ii. Non-ambulant children: regular passive or active assisted

stretching are necessary, both in lower and upper extremities. Night and

daytime AFOs should be supplied as well as KAFOs (not well tolerated by night)

in order to prevent painful contractures and foot deformity. Surgical intervention

is only recommended to alleviate specific symptoms, such as pain and

pressure, since it‟s generally ineffective as a routine procedure to correct

deformities “Muntoni et al. (2006); Kinali et al. (2007)”.

iii. Management of scoliosis: Scoliosis usually manifests after

loss of walking, shows rapid progression coincident with the pubertal growth

spurt and has a negative impact on respiratory function, feeding, seating and

comfort. It has been showed that daily steroid treatment along with the use of

KAFOs reduces the risk of scoliosis “Kinali et al. (2007)”. Surgery is usually

scheduled once the Cobb angle measured on scoliosis films is between 20-40º

but the optimal timing for surgical intervention is while lung function is

satisfactory and before cardiomyopathy becomes severe enough to risk

arrhythmia under anesthesia “Muntoni et al. (2006)”.

33

CONCLUSION

Research into therapeutic strategies for dystrophinopathies has

progressed rapidly over the past decade. The development of practical gene-

and cell-based therapies is an ongoing process that is still in its initial stages. It

is only through additional investigation that the drawbacks of these promising

approaches will be overcome and ultimately lead to the development of

therapeutic strategies effective in stemming the progressive and multifactorial

nature of DMD. Until these therapies become clinically available, supportive

pharmacological treatments are the main weapons against the disease‟s

progression with rehabilitation playing a core role in reducing morbidity,

increasing quality of life and prolonging lifespan of affected children.

Finally, it is probable that dystrophinopathies with such a complex

pathogenesis will only be defeated by a multidisciplinary approach aimed to

replace or correct the mutated gene product as well as counteract the

devastating consequences of the primary mutation on muscle structure and

function.

34

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