978 Current Pharmaceutical Design, 2010, 16, 978-987

Muscular Dystrophies: Histology, Immunohistochemistry, Molecular

Genetics and Management

Costanza Lamperti#,* and Maurizio Moggio°

#
UO Neurogenetica Molecolare, Fondazione IRCCS Isitituto Neurologico C. Besta, Via Celoria 11, 20100 Milano Italy, °UOS
Diagnostica Neuromuscoalre Fondazione Ospedale Maggiore Policlinico, Mangiagalli Regina Elena, IRCCS via F Sforza 34, 20122
Milano, Italy

Abstract: Muscle degeneration and regeneration are two of the most evident pathological events characterizing muscular diseases and in
particular muscular dystrophies. Muscular dystrophies are an heterogeneous group of hereditary diseases affecting both children and
adults, and are characterized by muscle wasting and weakness. Until now at least 30 different genes have been associated with muscular
dystrophies. They have been divided into several subgroups depending on the distribution of the muscle weakness. Thus, the
histopathological markers of all these forms are dystrophic changes at the muscle biopsy characterized by fiber size variability, fibres
necrosis, regeneration, inflammation and connective tissues deposition. As for now, no effective therapy is available for these diseases
but new inside has now been expanded in regenerative therapy such as cell therapy and gene therapy. This review is focused on muscular
dystrophies and new acknowledgments in regenerative therapy.
Keywords: Dystrophies, theraphy, exon skipping, LGMD, CMD.

INTRODUCTION present along with replacement of muscle by connective tissue and

Muscle degeneration and regeneration are two of the most
important pathomechanisms in muscle diseases and in particular
they are involved in the pathogenesis of muscular dystrophies.
Since now mutations in at least 30 different genes have been
associated with these diseases involving different proteins in the
muscle membrane and in the extracellular matrix. Whichever is the
protein involved, the “via ultima” is the dystrophic aspect of muscle
tissues, that is characterized by the degeneration of the muscle
fibres and a push to regeneration, inflammation and the substi-
tutions of contractile tissue with connective tissue that causes the
loss of muscle strength and muscle mass in patients. All these pro-
teins are part of the muscle membrane, such as dystrophyn,
sarcolgycans , caveolin, integrins and dysferlin, or part of the
extracellular matrix such as alpha dystrolgycan, merosins, collagen
VI or the nuclear envelop such as lamins and emerins. Never-
theless, although a large number of genes have been identified, the
mechanism leading to the degeneration and the regeneration of the
muscle fibres and the subsequent deposition of connective tissues,
is still not known. Muscular dystrophies are an heterogeneous
group of muscle diseases that are characterized by muscle weak-
ness, and depend on an alteration of specific muscle membrane or
extracellular matrix protein. Clinically, they are characterized by
progressive muscle wasting and weakness, with a wide clinical
heterogeneity: some of them have perinatal onset, while others
affect only adults. They could progress rapidly or be associated
with prolonged periods of stability; finally they could be associated
with multisystem involvement including cardiac and central
nervous systems. Usually patients become dependent to wheelchair
and death is caused by respiratory failure secondary to respiratory
and diaphragm muscle weakness. The biochemical tests show an
increase in the level of creatinkinase, even 10 times the normal
value, and the EMG usually shows myopathic change. The muscle
biopsy is essential to the diagnosis. As said before, all these
conditions share common histological features of ‘‘dystrophic’’
muscle biopsy changes, including fibre size variability, muscle fibre
degeneration and regeneration. Inflammatory changes may be
*Address correspondence to this author at the UO Neurogenetica
Molecolare, Fondazione IRCCS Isitituto Neurologico C. Besta, Via Celoria
11, 20100 Milano Italy; E-mail: Lamperti.c@istituto-besta.it
fat. The major advances over the last two decades due to the
identification of numerous sarcolemmal and extracellular matrix
(Fig. 1) proteins and associated genes, have improved diagnostic
precision by standardizing both the immunhistochemestry and the
genetic tests, in the view of genetic therapy. As for now, no therapy
has been identified but the potential role of promoting muscle
degeneration and restoring correct genes expression have been
studied. This review article is focused on muscular dystrophy
empathizing the diagnosis and new prospective in treatment.
MUSCULAR DYSTROPHIES
Muscular dystrophies are classified as Congenital Muscular
Dystrophies (CMD), dystrophinopathies, including Duchenne
Muscular Dystrophy (DMD) and Becker Muscular Dystrophy
(BMD), and Limb Girdle Muscular Dystrophies (LGMD).
Congenital Muscular Dystrophies
Congenital Muscular Dystrophies (CMD) are an heterogeneous
group of autosomal recessive disorders [1] presenting in infancy
and characterized by muscle weakness, contractures and dystrophic
changes at the skeletal muscle biopsy (Table 1). The association of
mental retardation with central nervous system (CNS) abnor-
malities, such as type II lyssencephaly, brainstem and cerebellar
alterations and eye abnormalities, is often present in Muscle Eye
Brain disease (MEB), Walker Warburg syndrome (WWS),
Fukuyama Congenital Muscular Dystrophy, or LAMA 2 deficiency.
These brain alterations can be secondary to an alteration of alpha
dystroglycan glycosylation during brain development. Recently, an
alteration of alpha dystroglycan glycosylation in brain has been
described in one patients with a CMD [2]. In some cases, as well as
in the Ullrich syndrome or in the less severe Bethlem syndrome, a
distal joint laxity can be the peculiar feature [3]. Instead, a milder
phenotype showing only muscle weakness is characteristic for the
CMD1C [1].
Creatine kinase (CK) could be markedly elevated or quite
normal. Nerve conduction studies may show demyelinating neuro-
pathy in merosin-deficient CMD [4] while electromyography
(EMG) is myopathic in quite all these forms [5] in which respira-
tory involvement is frequent and often is the dead cause. Muscle
biopsy shows typical dystrophic changes (degeneration and rege-

1381-6128/10 $55.00+.00 © 2010 Bentham Science Publishers Ltd.

Muscular Dystrophies
Fig. (1). Muscle fibers membrane.
neration of muscle fibres, and proliferation of fatty and connective
tissue). [1,5] Immunocytochemistry analysis Lamin and laminin, a-
dystroglycan, collagen VI, and
7 integrin lead to diagnosis of the
specific diseases.
These diseases can be classified into two major groups based on
the affected genes and the location of their expressed protein:
abnormalities of extracellular matrix proteins (LAMA2, COL6A1,
COL6A2, COL6A3), or abnormalities in the proteins involved in
the glycosylation of
dystroglycan, (fukutin, POMGnT1, POMT1,
POMT2, FKRP, LARGE) [6-8]. The latter mechanism is intriguing
and has aroused so great an interest in the last few years that this
condition is described as an identity apart called dystroglyca-
nopathy.
DG is a high glycosylated protein that forms a complex
with the membrane spannig Beta dystroglyca and conntecting the
extracellular matrix to the cytoskeleton [9,10] (Fig. 1). While
initially a clear correlation between gene defect and phenotype was
observed for each of these diseases, (Walker Warburg syndrome
was associated with mutations in POMT1 and POMT2, Fukuyama
congenital muscular dystrophy with fukutin mutations, and Muscle
Eye Brain disease associated with POMGnT1 mutations, have been
recently demonstrated that allelic mutations in each of these six
genes can result in a much wider spectrum of clinical conditions
[11] causing in some chase LGMD (see below). Thus, the crucial
aspect in determining the phenotypic severity is not to assess which
gene is primarily mutated, but how severely the mutation affects the
glycosylation of ADG [12] Although the incoming new scientific
development, at the moment about 65% of congenital muscular
dystrophies are without genetic diagnosis [13] and new entities
have been recently described with new different presentations [2-
Current Pharmaceutical Design, 2010, Vol. 16, No. 8 979
13] Only three patients right now has been described as associated
to mutations in the sarcolemmal membrane
1-7 integrin, on with
mental retardation associated with muscle weakness [6].
Finally, mutations in the nuclear involvement protein Lamin
A/C are undoubtedly those that can rise the most variable clinical
phenotype, ranging from a congenital muscular dystrophy with
contractures (Emery Dreifuss), to an isolated dilated cardio-
myopathy and a Charcot -Marie-.Toot disease type 2B1[14].
Dystrophinopathies
The dystrophin gene is the largest gene yet identified in
humans, identified for the first time in 1998. It is located in the
chromosome Xp21. [15] Its product, dystrophin, is postulated to be
essential for the protection of the sarcolemma from the stress of
repeated contractions by providing an indirect link between the
subsarcolemmal cytoskeletal actin and the intermediate filaments in
the muscle fibre with the extracellular matrix (Fig. 1). The muta-
tions in the dystrophin gene, cause the disturbance of the reading
frame, resulting in a severe reduction or absence of dystrophin in
the skeletal and cardiac muscle. Approximately 65% of patients
with DMD have intragenic out-of-frame deletions and approxi-
mately 10% of them have duplications of one or more exons of the
dystrophin gene. The remaining patients have point mutations or
other smaller gene rearrangements. Typically, out-of-frame
dystrophin gene mutations lead to a severe reduction or absence of
dystrophin in the muscle, resulting in DMD phenotype, whereas in-
frame mutations lead to the expression of a partly functional
truncated protein, resulting in the milder Becker muscular dys-
trophy (BMD) [16].
980 Current Pharmaceutical Design, 2010, Vol. 16, No. 8 Costanza and Moggio

Table 1. Clinical Feature and Genetic Classification of CMD. MDC: Congenital Muscular Dystrophy, WWS: Walker-Warburg
Syndrome, MEB: Muscle Eyes Brain Disease, CK: Creatine Kinase, RSMD Rigid Spine Muscular Dystrophy

Disease Protein Allelic Forms CNS Involvement Muscle/Respiratory Involvement

MDC1A Laminin-
2 LGMD1A White matter changes, neuronal Muscular dystrophy, respiratory insufficiency and
migration abnormalities and nocturnal hypoventilation, peripheral neuropathy
mental retardation (seizures)

MDC1B ? No involvement Facial weakness, diaphragmatic involvement with early

respiratory failure, rigid spine, secondary laminin
2
deficiency

Rigid Spine Syndrome Selenoprotein-1 No involvement Muscular dystrophy, axial hypotonia and weakness,
(RSMD1) lumbar scoliosis and cervical spine stiffness, respiratory
failure due to skeletal abnormalities and diaphragmatic
weakness, CK normal or mildly elevated

Integrin a7 Congenital Integrin
7 Mental retardation Mild muscular dystrophy/myopathy, torticollis, mildly
Myopathy elevated CK

Ullrich's Disease Collagen VI No involvement Neonatal muscle weakness, kyphosis of spine, joint
(1,2,3) contractures, torticollis, hip dislocation,
hyperextensibility of distal joints, cheloid formation,
invariable respiratory insufficiency, normal intelligence,

MDC1D LARGE Profound mental retardation, Muscular dystrophy

brain alterations

WWS POMT1 LGMD2K Type II lissencephaly/agyria, Muscular dystrophy, CK elevated

POMT2 hydrocephalus, eye abnormalities

MEB POMGnT1 LGMD2M Eye abnormalities and abnormal Muscular dystrophy, broad clinical phenotype,
neuronal migration significant hypotonia,

Fukuyama CMD Fukutin LGMD2L Severe mental retardation, Muscular dystrophy, cardiomyopathy.
seizures, eye abnormalities

MDC1C FKRP LGMD2I Structural brain changes, retinal Muscular dystrophy, congenital weakness and
changes with blindness hypotonia, calf and tongue hypertrophy, shoulder muscle
wasting, cardiomyopathy, CK elevated

DMD is the most common form of muscular dystrophy
affecting one in every 3500 live male births. Almost all boys with
DMD are symptomatic before the age of 5 years, with calf
hypertrophy, toe/waddling gait and Gowers’ sign. Subsequently,
they develop difficulties in running, jumping, and climbing steps.
Usually tendon retraction becomes evident in the first decade of life
associated with waddling gait, and lumbar lordosys. Independent
ambulation is lost between 10 and 14 years, with subsequent dete-
rioration in respiratory function and development of contractures
and scoliosis [17]. A cognitive evaluation carried on in twenty
DMD patients focused on memory, information processing/learning
ability, and executive functions, conclude that there is a worsening
in these patients compared to age matched control in the short term
memory and learning ability, while no significant difference was
evident in general intellectual ability [18].
BMD patients present symptoms similar to DMD but the
severity of the disease is variable presenting as a severe form DMD
like [19], a isolated dilated cardiomyopathy [20], a pseudometa-
bolic form characterized by muscle cramps with myoglobinuria
[21], a quadriceps myopathy [22]. Finally patients could be
completely asymptomatic showing only high serum CK levels. In
all the forms, the clues of the diagnosis is the calf hypertrophy that
is always present [19].
The severity of the disease is usually correlated with the amount
of remnant functional dystrophin. Patients with DMD have less
than 5% of the quantity of normal dystrophin. Patients with BMD
have at least 20% normal dystrophin levels [23]. Most patients with
BMD (85%) have dystrophin of abnormal molecular weight, often
with reduced dystrophin quantity. A minority have normal-sized
protein of reduced quantity. Because of this correlations between
Muscular Dystrophies
the amount of dystrophin and the clinical features, the study of
muscle biopsy is important in these diseases, and the genetic study
is not enough to give prognostic information to patients and their
families.
DMD/BMD carrier. Females may be asymptomatic or may
have mild muscle weakness with an increased CK, calf hyper-
trophy, myalgia, and cramps, and are at risk of dilated cardio-
myopathy [24]. Rarely, carrier female can present clinical mani-
festation similar to DMD patients. Approximately 50% of DMD
carrier females will have no family history of neuromuscular
disease [25]. The muscle biopsy could be normal or slightly patho-
logical with a mild increase of connective tissues. Dystrophin
immunocytochemistry in carrier or symptomatic females shows a
mosaic pattern, with a combination of fibres either containing or
lacking dystrophin. Quantitative genetic studies or sequencing may
then be confirmatory.
Before the age of 5 years, serum CK levels are 10 to 200 times
higher than normal levels, in boys with DMD and BMD, but
decline with advancing age. The CK does not discriminate between
dystrophinopathies. Nerve conduction studies are normal.
Electromyography shows myopathic changes. The muscle biopsy is
the main instrument to make diagnosis of DMD and BMD.
Classically, it shows degeneration, regeneration, and variability of
fibre size with replacement of muscle by fat and connective tissue.
Milder changes are seen in BMD. Immunocytochemical studies are
fundamental to differentiate DMD to BMD. In DMD a complete
absence of binding with the antibodies for the COOH, NH2 and
Rod domain is observed. A partial reduction or the presence of a
truncated proteins lead to the diagnosis of BMD. The immunbolt is
a more sensitive test, and it has to be used to confirm the diagnosis.
Genetic testing techniques include the polymerase chain reaction
(PCR), western blotting, mutation scanning, and/or sequence ana-
lysis [17] .The DNA extracted from whole blood or isolated white
cells can be used for diagnosis. Prenatal diagnosis is routinarily
done in DMD,BMD carrier . Recently the MLPA system has been
identified as useful to rapidly identify duplication and deletion in
the dystrophin gene [26]. Pulmonary function studies, electrocar-
diography, and echocardiography should be performed annually
after 10 years old.
Limb-Girdle Muscular Dystrophies
The Limb Girdle Muscular Dystrophies (LGMD) are a hetero-
geneous group of inherited progressive muscle disorders affecting
predominantly shoulder and pelvic girdle muscles. There are at
least eighteen different subtypes of LGMD, seven with an auto-
somal dominant ineritans (LGMD1A-G) and eleven with an
autosomal recessive pattern of inheritance (2A-K) (Table 2) [27-
29].
AD-LGMD
The autosomal dominant forms (LGMD1) represent probably
less than 10% of all LGMD cases. The proteins involved in AD
LGMD are structural proteins such as myotilin, a component of the
sarcomeres responsible for the LGMD1A, Lamina A/C, part of the
nuclear envelop for the LGMD1B, and Caveolin (LGMD1C),
involved in the constitution of caveole in the muscle membrane
(Fig. 1). For the LGMD 1D-G all loci have been identified by
linkage analysis, but the genes involved are still unknown [29]. The
onset of the disease is variable ranging from infancy to 2nd-3rd
decade of life.
AR- LGMD
All mapped genes responsible for autosomal recessive forms
(LGMD2) have been identified. They encode highly different
proteins involved in all aspects of muscle cell biology, such as the
sarcolemmal muscle membrane (sarcoglycans, dysferlin), the
sarcomere (telethonin, titin), the muscle cytosol (calpain-3,
Current Pharmaceutical Design, 2010, Vol. 16, No. 8 981
TRIM32), and the glycosylation pathway enzymes (Fukutin Related
Protein, Protein O-Mannosyltransferase 2) [30-33].
Clinically, these muscular dystrophies present with progressive
muscle weakness and atrophy with predominant involvement of
scapular and pelvic girdles. The neck flexors and extensors may be
involved and typically facial muscles are not involved and
cognition is usually normal. Cardiac or other systemic involvement
is variable and can be present in LGMD 1B, 1D, 2C-G, and 2I.
Typically elevated serum creatine kinase (CK) levels are present.
Nevertheless, LGMDs usually present a large clinical variability
regarding age of onset, patterns of skeletal muscle distribution,
heart damage, respiratory involvement and rate of progression that
result in variable morbidity and disability. In some cases such as
caveolinopathy, a rippling phenomena, cramps and calf hypertrophy
are the main features [34]. Nevertheless, calf hypertrophy is com-
mon also in LGMD2I [17]. Serum CK is usually modestly elevated
but can be very high in the sarcoglycanopathies, dysferlinopathy,
and caveolinopathy. The autosomal recessive LGMDs generally
have an earlier onset, more rapid progression and higher serum CK
values than the dominant LGMDs. EMG shows myopathic changes
and is particularly useful in discriminating mild forms of dominant
LGMD, which may be associated with only very mild serum CK
elevation, from spinal muscular atrophy type II and chronic
inflammatory neuropathies. The muscle biopsy still represents an
important step in the diagnostic process.
Muscle biopsy could range from normal biopsy to a severe
dystrophic pattern. In dystrophies such as dysferlynomathy and
calpainopathy, a severe inflammation is often present and it is
characterized by large infiltrates and several necrosis, while these
aspects are really rare in caveolinopathy [30]. Moreover, in dysfer-
linopathy, several fibres in regeneration are present, indicating that
the dysferlin protein may be involved in the regenerating process,
by controlling cytokine expression [27]. Some vacuoles could be
present in the muscle biopsy in LGMD1A. The immunostaing for
the sarcoglycan, dysferlyn, caveolin and alpha dystroglycan is
normally used for the diagnosis. Recently the immunostaing for
calpain 3 has been found useful for the diagnosis in association with
the immunoblot for the specific proteins [30-31]. However, protein
deficiency documented by immunohistochemistry in muscle may be
secondary and in most patients a definite diagnosis can be obtained
only by genetic analysis, as the genetic test confirms the diagnosis.
Treatment is symptomatic.
General Management
A specific therapy for muscular dystrophy is not available, thus
considering the possible role of muscle inflammation in the patho-
genesis of the disease, the corticosteroid is the main drug used in
dystrophic patients and in particular in DMD. This therapy is
associated with physiotherapy and orthesis management of heart
and respiratory complications, scoliosis and nutritional aspects.
Randomized controlled clinical trials of daily oral prednisone
demonstrated an improvement in muscle strength and function in
children with DMD, compared to baseline and to controls [35,36].
In these trials, a dose of 0.75 mg/kg/day of prednisone was used
[37,38]. Long-term neuromuscular benefits of corticosteroid use
include prolonged independent ambulation and a lower prevalence
of scoliosis [39-41]. Adverse effects of prednisone, in particular
related to behaviour, weight, and bone density often necessitate the
reduction of the dose over time.
The earliest sign of DMD is a progressive decline in limb
muscles function. This is primarily due to skeletal muscle weakness
and secondarily to joint contractures. Physiotherapy is the primary
therapy to prevent joint deformities 9. In DMD, rehabilitation in
knee, ankle, foot orthoses (KAFOs) at the time of loss of inde-
pendent walking is effective in prolonging walking for an average
of 18 months to 2 years. This has been associated with reduced
incidence of scoliosis.
982 Current Pharmaceutical Design, 2010, Vol. 16, No. 8 Costanza and Moggio

Table 2. Clinical Features and Genetic Classification of LGMD. LGMD: Limb Girdle Muscular Dystrophy, AR: Autosomal
Recessive, AD: Autosomal Dominant, AO: Age of Onset, IH, Immunohistochemistry DG: Dystroglycan

LGMD HE Protein AO Clinical Manifestations Muscle Biopsies Cardiac Involvement

LMGD 1A AD Myotilin II-III Dysfagia and myopathy Dystrophic changes

Vacuoles, accumulation of myotilin at IH

LMGD 1B AD Laminin A/C I-III Myopathy, CMT2, congenital Dystrophic changes Common
myopathy.

LMGD 1C AD Caveolin 3 I-IV Calf hypertrophy, Rippling, High variability in fibers size, absence of Un common
muscle weakness caveolin at IH

LMGD 2A AR Calpain 3 I-IV Posterior compartments of the Dystrophic changes, regeneration, Un common
limbs reduction of calpain 3 at WB

LMGD 2B AR Dysferlin I-IV Distal myopathy. Dystrophic changes , inflammatory cells , Un common
reduction of dysferlin at WB

LMGD 2C-D- AR Sarcoglycan
 I-II DMB like, BMD Like, calf Dystrophic changes, reduction of one or Common
E-F hypertrophy all sarcoglycans at IH

LMGD 2G AR Telethonin I-II Calf hypertrophy or hypotrophy, Dystrophic changes, reduction of Un common
distal leg weakness Telethonin at IH

LMGD 2H AR TRIM32 I-IV Possible mild facial weakness Dystrophic changes

LMGD 2I AR FKRP I-IV Calf hypotrophy, myopathy, Dystrophic changes and reduction of
Common
muscle pain DG

LGMD 2J AR Titin I-II Distal weakness, proximal distal Dystrophic changes Common
myopathy

Respiratory and cardiac care are two aspects that have to be
monitored. In the past, the onset of symptomatic sleep hypoven-
tilation signified imminent demise, as the only way to prolong life
was mechanical ventilation through tracheotomy. The greatest
contribution to improve the respiratory care, leading to extend the
longevity in DMD, is the domiciliary non-invasive ventilation in
1990 [42-44]. Moreover, it seems that long term steroid therapy
would improve pulmonary function.
Dilated cardiomyopathy is sometimes associated with muscular
dystrophy causing ventricular dysfunction, defined as a shortening
fraction, less than 28%. Nevertheless, corticosteroid treatment is
protective against this dysfunction, if it starts prior to cardiac
involvement [45,46].
Early angiotensin-converting enzyme inhibitor (ACEi) treat-
ment may also be indicated in DMD as suggested by a trial of
perindopriol, although some cardiologists do not find any treatment
necessary for complication that are often asymptomatic for a long
time [47]. There are prospective trials of ACEi and angiotensin II-
type 1 receptor blockers in patients with dilated cardiomyopathy
from a variety of aetiologies, indicating improvement both in
survival and in symptoms [48-50]. Such studies argue for early and
continued use of corticosteroids and ACEi/angiotensin II-type 1
receptor blockers in patients with DMD.
NEW PROSPECTIVE IN THERAPY
Muscle weakness in muscular dystrophy is secondary to muscle
degeneration due to a mutations in genes composing the DGC
(dystrophyn glycoprotein complex). Mutation in one of these genes
affects proteins that form a link between the cytoskeleton and the
basal lamina. Absence of one of these proteins often causes the
disassembly of the whole multiprotein complex associated with
dystrophin, leading to increased fragility of the sarcolemma,
especially during intense contractile activity. This results in an
increased calcium entry and focal or diffuse damage to the fibre
[51]. Damaged or dead fibres can be repaired or replaced by
satellite cells [52]. These cells are actually considered the resident
‘stem-like’ cells in skeletal muscle. They are responsible for muscle
growth and regeneration in postnatal life [53]. However, dystrophic
satellite cells share the same molecular defect and produce fibres
that are also prone to degeneration. With time, the population of
satellite cells is exhausted and the muscle tissue is progressively
replaced by connective and adipose tissue. Moreover, the death of
muscle cells provokes a secondary inflammatory reaction inducing
a further muscle damage.
Thus, as said before, there is not a cure for these diseases,
therefore there is a great interest in developing new promising
strategies and therapies. One of these strategies consists in the
tentative restoration of the normal proteins with the aim of
correcting the gene defect and the regenerations of muscle tissues
using stem cells or factors that promote regenerations. Each stra-
tegy has both advantages and limitations: for example, strategies
that repair the dystrophin gene might be suitable only for a subset
of mutations, whereas cell and gene therapies are limited by costs
related to cell or vector production, and they can be available only
for a limited number of patients [54]. These strategies are: 1 gene
therapy, 2 cell therapy, 3 drug therapy
Gene Therapy
Exon Skipping
As previously described, muscular dystrophies are secondary to
mutations in genes encoding for proteins of the DGC. In particular,
it is well known that either the deletions or the mutations in the
dystrophin gene produce an alteration of the reading frame of the
gene. One therapeutic strategy is the use of an antisense oligo-
Muscular Dystrophies
nucleotide (OAs) to modify the dystrophin mRNA splicing. The
OAs prevent the normal splicing of the gene, by masking crucial
areas of the messenger RNA during the splicing process that
produce an “exon skipping “. The skipping leads to the restoration
of the reading frame of the mRNA and that produces (producing) a
phonotype similar to the milder BMD and provides significant
clinical improvement in DMD patients. It has been estimated that
the skipping of 12 exons around exon 51 could be therapeutic for
about 20 % of dystrophin deletions [55]. An open trial on four
patients affected with DMD using a 2-O-methyl phosphorothioate
antisense oligonucleotide, demonstrated the skipping of exon 51
and the sarcolemmal dystrophin expression 28 days after single
injection into the tibialis anterior muscle [56]. The amount of
dystrophin produced ranged from 3% to 12% of control specimens
by Western blot and from 17% to 35% of control specimens by
ratio of dystrophin to laminin _2 considering the immunohisto-
chemistry enhance expression [56]. Recently, a single-blind
placebo-controlled dose-escalation study in seven patients with
DMD, was carried out to assess the safety and biochemical efficacy
of an intramuscular morpholino splice-switching oligonucleotide
(AVI-4658) that skips exon 51 in dystrophin mRNA. The intra-
muscular injection in the extensor digitorum brevis (EDB), and the
contralateral muscle have been taken as a control. This trial showed
that the intramuscular AVI-4658 was safe, and induced the local
expression of dystrophin within treated muscles. This proof-of-
concept study could be the starting point to a systemic clinical trial
[57].
Nonsense Suppression
About the 15% of DMD mutations are stoop codon point muta-
tions that produce a truncated or absent protein. Aminoglycosides
cause misreading of the RNA code at the premature but not at the
normal termination codons, leading to the insertion of alternative
amino acids at the site of the mutated codon, transcription and
protein formation. Gentamicin has been found to be effective in
mdx mice in some cases [58], whereas it is ineffective in other
cases, (it is ineffective) [59]. Moreover, gentamicin presents a lot of
side effect in long term administration. A similar molecule,
PTC124, is now available. This molecule specifically allows to
read-trough the nonsense mutations, without any alteration on
normal translational stop signs. PTC 124 was well tolerated in
phase 1 studies and in 62 healthy volunteers [60]. A phase 2A open
label trial is currently ongoing, and will evaluate both muscle
strength and dystrophin expression, and serum CK levels. A
randomized, controlled 48-week treatment phase 2B trial in DMD
and BMD patients with nonsense mutations has begun, being the
total distance covered during a 6-minute walking test the primary
outcome measure (NCT00592553, clinicaltrials.gov). This study
has been recently extended to other 96 weeks.
AVV Mediated Gene Transfer
The third therapeutic possibility to restore the dystrophin in the
muscle plasma membrane is represented by the adenoviral vector
(AVV). The AVV is the leading viral vector for human gene
therapy, but it has a maximum package of 5 kb, while the dys-
trophin gene is about 11 kb. For this reason, a truncated dystrophin
gene construction has been provided, and it is called mini or micro-
dystrophin. Mini-dystrophins and micro-dystrophins have been
shown to ameliorate the dystrophic pathology of the mdx mouse
[61-63]. This strategy allows to express a truncated but partly
effective dystrophin protein in muscle, thus the adminis-tration of a
viral agent could produces an immune response to the vector. A
phase I trial of a micro-dystrophin under a cytomegalo-virus
promoter in a modified AAV is now underway in boys with DMD
(NCT00428935, clinicaltrials. gov). A phase I study has started
now and will provide useful information about safety, and the
Current Pharmaceutical Design, 2010, Vol. 16, No. 8 983
secondary outcome is the measurement of the mini-dystrophin gene
expression at the site of gene transfer and the muscle strength
evaluation by Maximal Volume Isometric Contraction Testing.
Cell Therapy
Cell-based therapy is a potential solution toward restoring
dystrophin expression in skeletal muscles. This therapy exploits the
ability of wild-type muscle precursor cells to fuse with damaged
DMD myofibers, thereby introducing nuclei that express the normal
dystrophin gene in the muscle syncytia. Several adult stem cell have
been isolated, and are characterized and used in animal trans-
plantation experiments [64]. A few examples of stem cells showing
a potential therapeutic affecting or used in animal clinical trial are
listed below.
Muscle Derived Stem Cell (MDSC)
Muscle satellite cells play a central role in postnatal muscle
growth and regeneration, and are promising tool for cell therapy.
Satellite cells are dormant progenitors located at the periphery of
skeletal myofibers that can be triggered to proliferate for both self-
renewal and differentiation into myogenic cells secondary to
specific stimuli such as oxidative stress [65] were able to directly
isolate a pure population of satellite cells, injecting them into
dystrophic dogs and restoring dystrophin expression 3 weeks post-
transplantation. Into irradiated dystrophic mice, they also formed a
small amount of the satellite cell pool that expressed both Pax7 and
Pax3 [65]. The results obtained in the mouse model led to test the
myoblast injection in DMD patients in phase I clinical trials.
Unfortunately, these trials demonstrated that myoblast transplan-
tation is an inefficient technique: the efficiency of the dystrophin
production in muscle fibres of DMD patients was very low ( 1%)
and there was no functional or clinical improvement in the children
[66].
Mesoanguiblast
Mesoangioblasts in mice and dogs and pericyte in human are
multipotent progenitors of mesodermal tissues, physically
associated with the embryonic dorsal aorta in avian and mammalian
species. Cossu, et al in 2003 demonstrated the capacity of
mesoangioblasts to differentiate in various mesodermal phenotypes
qualifying these progenitors as a novel class of stem cells. Systemic
delivery in dystrophic mice and dogs has been performed [67]. The
intra-arterial transplantation of donor mesoangioblasts ameliorated
defective muscle structure and function in dystrophic mice [68] and
in cyclosporine immunosoppressed dogs [69]. In particular, Cossu,
et al. showed that dogs treated with mesoangioblasts had an
extensive expression of dystrophin in the majority of the muscle
fibres and were characterised by both normal force contraction and
amelioration of defective mobility [69]. Recently, similar results
were achieved with mesoangioblasts transplanted into mdx/utrophin
null mice [70]. Because of these encouraging results, a clinical trial,
that uses donor stem cells from an HLA (human leukocyte antigen)
identical donor have been planed. However, this study, at least in
dogs, do not provide control animals treated only with cyclosporin
A (CSA) [70,71]. The possible beneficial effect of CSA in muscular
dystrophy is currently being tested in a specific clinical trial in
Germany [72]. Nevertheless, one paper reports beneficial effects of
the drug in dystrophic mice whereas other papers clearly show a
deleterious long-term effect, resulting in the inhibition by CSA of
the calcineurin pathway that is essential for muscle regeneration
[73-75]. A study in dogs involving a restricted number of animals
showed a great variability in the progression of the disease. For all
these reasons, a great prudence has to be used before treating
patients. No adverse events related to mesoangioblast infusion have
been observed in a total of 16 dogs treated [69] or currently under
treatment, and a group in Italy is going to perform a clinical trial in
DMD patients.
984 Current Pharmaceutical Design, 2010, Vol. 16, No. 8
Circulating Stam Cell
Several studies have demonstrated that wild-type (wt) total
bone marrow-derived or side population (BM-SP) cells are
incorporated into regenerating skeletal muscle fibres when
transplanted into dystrophic mice [76-78]. However, in some cases
transplanted cells failed to restore the expression of that protein,
suggesting that under standard conditions they have little
therapeutic potencial [79,80]. In other cases, results have been more
encouraging. For example, bone marrow mesenchymal stem cells,
which show little myogenic differentiation, became highly
myogenic when engineered to express high levels of intracellular
Notch protein, and produced dystrophin in many fibres when
transplanted into mdx mice [77]. Another cell population, isolated
from blood and from skeletal muscl expressing the stem cell CD133
antigen, has been shown to give rise to dystrophin- positive fibers
when transplanted into scid (Severe Combined Immuno-
deficiency)/mdx mice [76]. Torrente et colleagues, demonstrated
that this cells can be injected safely in DMD patients not only
without side effects but also promoting an increase in the number of
capillaries per muscle fibres [81], and that DMD CD133+ cells can
be genetically modified to re-express a functional dystrophy [82].
Future clinical trials with this cell types are now feasible.
Drug Theraphy
The last strategy is to stimulate growth and regeneration of
dystrophic muscle or to inhibit the inflammation by promoting
muscle degenerations. These strategies may provide functional
benefits by increasing size and strength of minimally affected
muscle, or by improving the quality of the composition of
dystrophic muscle. Thus, they will not be a cure for the diseases, in
fact they do not address the pathophysiology of the single disease,
such as the loss of dystrophin or sarcoglycans, but it can produce
benefits in all the dystrophic muscles, as it is focused on the
pathomechanism of the dystrophic changes. Postnatal growth and
regeneration are regulated by a variety of endogenous growth
factors, such as members of the transforming growth factor-
.
Myostatin
Myostatin is a member of the TGF-
superfamily and is an
endogenous negative regulator of muscle growth. [83]. Loss of
function mutations in myostatin results in massive muscle
hypertrophy and hyperplasia with approximately doubling of
muscle mass in humans, as described in one patient with a loss of
function mutation in the myostatin gene [84,85]. Because myostatin
is a negative regulator of muscle mass, several studies have been
performed to investigate a potential therapeutic effect of antago-
nizing the myostatin pathway. Myostatin can be inhibited post-natal
by a variety of mechanisms that similarly induce muscle growth
[86]. Inhibition of myostatin with a neutralizing antibody and with a
modified myostatin pro-peptide, delivered via AAV, ameliorates
the dystrophic phenotype of the mdx mouse model [87,88]. Based
on these results Wyeth Pharmaceuticals (http: //www. wyeth.com/)
developed neutralizing antibodies among which MYO-029 has
entered in clinical trials for different forms of muscular dystrophy,
in particular BMD. No severe adverse events were observed in the
short term treatment except hypersensitivity to the antibody in some
patients. The first clinical trial of a myostatin inhibitor in a disease
population was recently completed demons-trating safety without
target-related side effects of a neutralizing antibody to myostatin,
MYO-029 in subjects with diverse adult muscular dystrophies,
including Becker muscular dystrophy [89]. Long-term safety and
efficacy are currently being tested. Further information is available
on the website of the Muscular Dystrophy Association (http:
//www.mdausa.org). Improvement in muscle strength and function
were not demonstrated in this trial, while biological activity of
MYO-029 was suggested by trends in increased muscle mass and
fibres diameters [89]. In the future, this therapy could be combined
with gene or cell therapy. Recently, some authors demonstrated that
Costanza and Moggio
myostatin inhibition by follistatin, which opposes the activity of
myostatin mentioned above, is a good method to improve myoblast
transplantation and muscle function. [90].
TGF-
1
TGF-B1 is another member of the TGF-
superfamily. It is
homologous to myostatin, and induces many similar effects on
muscle, including the inhibition of proliferation, differentiation of
muscle precursor cells and stimulating the fibrosis [91-93].
Contrary to myostatin, TGF-
1 is up-regulated in early DMD [94-
96]. These features would suggest that the inhibition of TGF-
1
would be a possible therapeutic strategy. However, TGF
1 is
widely expressed and controls a range of processes in various
tissues throughout the body, raising concerns of specificity and
toxicity. Thus, some studies have showed that losartan, a
angiotensin II-type1 receptor bloker usually used for hypertension,
is well tolerated in mdx mice mdx mice treated for 6 to 9 months
with this drug demonstrated less fibre size heterogeneity, less
fibrosis per area, and increased strength by grip strength and
absolute force of explanted muscle [97]. Many older DMD patients
are currently being treated with losartan for the presumptive cardiac
benefits of angiotensin II-type 1 receptor blockers.
CONCLUSION
Muscular dystrophies are severe progressive muscle diseases
involving both adults and children, characterized by the degene-
ration of muscle tissues and substitution of muscle fibres with
connective tissues. The supporting therapy such as corticosteroids,
phisiokinesitherapy, hart and non invasive ventilatory support are
useful tools to assist these patients and arise the possibility of a
better quality of life and a longer life expectancy for these patients.
Although a therapy for these diseases has not been identified,
multiple novel therapeutic agents have recently entered clinical
trials or are expected to in the near future. The therapy strategies
involve gene expression modulations, cell therapy and the
stimulation of regenerating factor. It is unlike that any of the current
approaches will independently cure this devastating disease, but it
is possible that the combination of more agents could provide a
therapeutic approach. Nevertheless, great part of the pathogenesis
and the pathomechanism of these diseases remains to be under-
stood. Even if, since 1993, 600 papers about exon skipping and 140
papers in stem cells were published, and right now there are
numerous trial in animals and humans being performed all the
world, a useful therapy is still unknown. In our opinion the effective
pharmacological agent or a combination of strategies might be
found when more will be understood about the pathophysiology of
these disorders.
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