Abstracts from 11th International Congress of World Muscle Society - Bruges, Belgium, October 2006
1) A phase I/II clinical trial in Duchenne muscular dystrophy using IM and IV delivered antisense oligonucleotides: The MDEX consortium
V.
Arechavala, K. Bushby, G. Dickson, I. Graham, M. Kinali, K. Liu, J. Morgan, F.
Muntoni, L. Popplewell, T. Partridge, F. Thorogood, K. Wells, N. Wells, M. Wood
and H. Yin – UK
Duchenne muscular dystrophy (DMD) is the most common childhood form of
muscular dystrophy, and out-of-frame deletions and duplications the most common
type of mutations. Antisense oligonucleotides (AO) can induce exon skipping and
restore the reading frame in animal models and cultured DMD cells. We are
involved in a Department of Health (DOH) funded phase I/II clinical trial which
first stage it to administer intramuscularly AO in DMD children with deletions
which can be rescued by skipping of exon 51. During the first year of this 4
year project we have performed preparatory clinico-pathological studies to
establish the grade of preservation of distal leg muscles and the frequency of
revertant fibres in non ambulant DMD children, using both MRI and biopsies taken
at the time of planned surgery, as we will need to target this age range in the
initial IM studies. We have established a biobank of cell cultures from DMD
patients with a variety of deletions to validate AO in the human. From a
preclinical point of view, we have optimised sequence targets, by testing eight
different exon 51 AO, which eventually have allowed us to choose an optimal
sequence. We have chosen to use a morpholino backbone AO for the clinical trial.
We have also developed a transgenic approach using a luciferase reporter gene to
facilitate the study of systemically delivered AO. We expect to start the IM arm
of the trial in exon 51 deleted DMD boys by the end of 2006 and to be able to
analyse the data by summer of 2007. If this approach will be found to be safe
and able to restore dystrophin production, we plan to commence a systemic (I.V.)
delivery trial with exon 51 by the end of 2007. We wish to thank Dr. J.Versnel,
Study Coordinator, and the Muscular Dystrophy Campaign, The DMD Parent Project
UK, the DMD Family Support group and the Department of Health UK for financial
support. We thank Prof. Steve Wilton, Perth, Australia for the generous sharing
of AOs sequence and advise, and Dr. J. van Deutekom, Leiden, The Netherlands,
for collaborative studies aimed at validating the different AO in the hmDMD
mouse.
2) Gene expression profiling to monitor therapeutic and adverse effects of antisense-induced exon skipping for Duchenne muscular dystrophy
P.A.C. ’t Hoen, C.G.C. van der
Wees, A. Aartsma-Rus, R.
Turk,
A. Goyenvalle, O. Danos,
L. Garcia, G.J.B. van Ommen,
J.C.T. van Deutekom
and J.T. den Dunnen
- Nederland and France
The objective of our study was to assess the utility of genome-wide gene
expression profiling for the preclinical evaluation of the efficacy and safety
of disease treatment. As a model we used antisense oligonucleotide (AON) induced
exon skipping for Duchenne muscular dystrophy (DMD). Muscles from mdx mice, a
well-characterized animal model for DMD, were injected with antisense constructs
that restore the open reading frame in the Dmd gene. Both synthetic AONs
complexed with different carriers to enhance cellular uptake and recombinant
adenoassociated virus (rAAV) expressed antisense sequences were evaluated. At
different time points after treatment muscle RNA was isolated and gene
expression profiles were generated using oligonucleotide microarrays.
Polyethylenimine (PEI)-complexed AONs restored the reading frame slightly more
effectively than uncomplexed, F127- or Optison-complexed AONs. However, PEI
induced the expression of many immune genes, reflecting an aggravation of the
inflammation present in untreated mdx mice. Expression profiles in Optison and
F127-injected muscles were similar to those of saline-treated muscles, implying
that these carriers did not evoke adverse responses. Due to the moderate levels
of exon skipping obtained in these experiments, a significant shift towards
wild-type expression levels could not be detected. Injection with rAAV vectors
resulted in much higher skipping levels, concomitant production of dystrophin
and a greatly improved histological appearance of the muscle. When the exon
skipping levels increased, the expression of genes previously shown to be
elevated in muscular dystrophies, partly or completely returned to wild-type
expression levels. Reductions in inflammation and fibrosis were among the most
prominent changes observed. Our results show that genome-wide gene expression
profiling is a powerful tool for the evaluation of both desired and adverse
effects of new pharmacological therapies. It is sensitive and can detect changes
that are not yet visible histologically. In addition, its ability to
simultaneously monitor a large number of different biological processes may
assist in the early detection of potential undesired side effects, thereby
reducing overall development time and cost considerable
3) Towards safe and efficient full-body delivery of antisense oligonucleotides for the treatment of Duchenne muscular dystrophy
J.A.
Heemskerk,
C.L. de
Winter, N. Heuvelmans, P. van Kuik, S. de Kimpe, G. Platenbur and J.C.T. van
Deutekom - Netherland
Duchenne muscular dystrophy (DMD) is caused by frame-shifting mutations in the
DMD gene that lead to dysfunctional truncated dystrophin proteins. By inducing
the specific skipping of exons from the pre-mRNA, the reading frame and thus the
dystrophin expression can be restored. The resulting proteins carry an internal
deletion, but include the essential C-terminal parts, comparable to the
partly-functional dystrophins found in the mostly milder Becker muscular
dystrophy patients. Exon skipping can be induced with antisense oligonucleotides
(AONs) targeting sequences involved in the recognition of exons by the splicing
machinery. We have shown that this strategy has therapeutic potential in several
patient cell cultures and in the mdx mouse model after intramuscular injections.
Our current focus is to find the best AON formulation, dose, regimen, and
administration method to deliver AONs safely and efficiently to muscle cells
throughout the body. In this study, AON (2′-O methyl PS RNA) uptake in
mdx and wild type mice was compared. In addition, different injection sites
(intravenous, subcutaneous and intraperitoneal) were evaluated. An AON-specific
ELISA showed that AON uptake by muscle fibres was approximately four times
higher in mdx mice than in wild type mice, while uptake was equal in other
organs. This implies that the damage of the muscle fibre membrane in mdx mice
indeed enhances AON uptake. It was further shown that the uptake was highest
after IV injection, followed by SC injection and finally IP injection. The
levels of AON uptake corresponded to the levels of exon skipping as determined
by RT-PCR analysis; the highest skipping levels were observed after IV injection
(over 40% in various skeletal muscle groups, 30% in the diaphragm and 5% in the
heart). This resulted in significant amounts of dystrophin, even in the heart.
Serum creatine kinase levels were decreased when compared to placebo treated
littermates, and muscle endurance experiments with a RotaRod measuring device
showed a clear improvement.
4) Antisense oligonucleotide design for therapeutic antisense-mediated exon skipping for Duchenne muscular dystrophy
A. Aartsma-Rus, C.L. de Winter,
W.E. Kaman, A. A.M. Janson
and J.C.T. van Deutekom - Netherlands
Both the severe Duchenne and the milder Becker muscular dystrophies (DMD and BMD,
respectively) are caused by mutations in the dystrophin gene. We are pursuing a
therapy that aims to restore the open reading frame in DMD transcripts. Using
antisense oligoribonucleotides (AONs) the pre-mRNA splicing is modulated such
that one or more exons are skipped, thus allowing reading frame restoration and
the generation of BMD-like proteins. We have so far designed 123 AONs that
target 39 different DMD exons; 81 of these are effective at variable levels. The
AONs are complementary to exon-internal sequences and are mainly directed at
open structures within the target RNA (as predicted by m-fold). It is thought
that the majority of exons contains exonic splicing enhancer sites (ESEs). The
splicing factor family of SR proteins binds to these sites and thus facilitates
exon inclusion by recruiting other splicing factors such as U1 snRNP to the
splice sites. Retrospective analysis of our AON series with ESE predicting
software revealed that effective AONs target significantly more putative ESEs
than ineffective AONs. As for some exons the skipping levels are low, we
hypothesized that for these exons two mutually exclusive ESEs might be present.
Indeed using a combination of AONs within a single exon induced the skipping of
thus far ’unskippable’ exons and improved exon skipping levels for inefficiently
skipped exons. These results indicate that exon-internal AONs are likely to act
by steric hindrance of SR protein binding to ESEs and that we can thus use ESE
predicting software to fine-tune the design of new AONs.
5) Improvement of muscle mass after injection of AAV vectors expressing either myostatin shRNA or Activin receptor IIb shRNA
J. Dumonceaux, S. Marie and L.
Garcia - France
Duchenne muscular dystrophy (DMD) is a severe degenerative disorder of skeletal
and cardiac muscle. DMD patients show a progressive muscle weakness which begins
in early childhood. Our goal is to oppose this continuous muscle wasting by
altering the myostatin, a negative regulator of skeletal muscle mass. This
protein binds to its cell-surface receptor (Activin receptor IIb, AcvRIIb) to
inhibit both proliferation and differentiation of myoblasts. Indeed, it has been
shown that myostatin deficient mice have a double mass phenotype compared to
wild type animals. Our hypothesis is that inactivation of myostatin function may
rescue muscle wasting in DMD patients as well as in its murine model, the mdx
mouse. We investigated RNA interference strategy to inhibit functions of
myostatin. We have decided to target both AcvRIIb and myostatin. Mice were first
injected intra muscularly using an AAV vector expressing either the AcvRIIb
shRNA or myostatin shRNA. Preliminary results show that a 50% down regulation of
AcvRIIb mRNA in the TA of mdx mice results in a 15% increase in muscle mass. We
also show that myostatin shRNA interfered with 95% of myostatin mRNA expression.
In addition, when mice were injected systemically with this AAV expressing
myostatin shRNA, we observed, 25 days after injection, that the body weight of
the injected group is 15% more important than the non-injected group, suggesting
a body-wide delivery of the vector. In conclusion, we show the possibility to
manipulate muscle mass in dystrophic mice by interfering with the expression of
myostatin activities. We are now investigated the possibility to combine
myostatin inhibition and dystrophin rescue in a large animal model of DMD.
6) Autologous transplantation of muscle-derived AC133+ stem cells
M. Belicchi, C. Marchesi,
R. Benchaouir, G. D’Antona, V. Mouly, G. Buttler-Browne, G. Cossu, N. Bresolin,
L. Garcia
and Y. Torrente -
Italy and France
We recently reported that human circulating AC133+ progenitor cells can be
induced to differentiate into skeletal muscles of scid-mdx dystrophic mice.
However, in blood specimens of dystrophic patients there are low numbers of
identifiable AC133+ stem cells that are difficult to be expanded in vitro. Here,
we show that autologous transplantation of muscle-derived AC133+ cells in
Duchenne muscles is safety and feasible. No local or systemic side effects were
observed in treated DMD patients included in a randomised double blinded phase I
study. Surprisingly, two treated patients had an increased ratio of capillary
per muscle fibers with a switch from slow to fast myosin+ myofibers and partial
recovery of some contractility force as showed by functional studies. We thus
extended these clinical observations combining gene and stem cell treatment by
the genetic engineering of AC133+ stem cells using exon-skipping approach which
rescue the dystrophin expression. In these experiments the intramuscular and
intra-arterial transplantation of engineered DMD stem cells caused a significant
amelioration of skeletal muscle structure and function when delivered to
scid-mdx dystrophic mice. We speculate that transplantation of DMD engineered
AC133 positive stem cells could represent a future treatment for Duchenne
muscular dystrophy.
7) NT-proBNP is not associated with dilated cardiomyopathy in Becker and Duchenne muscular dystrophies
S.M. Schade
van Westrum, L. Dekker, E. Endert,
R.J. de Haan,
A. A.M. Wilde,
M. de Visser
and A.J. van der Kooi -
Netherlands
N-terminal proBrain natriuretic peptide (NT-proBNP) is a stable protein produced
by myocytes of mainly the left cardiac ventricle as a response to stretch. It is
increasingly used as predictor for the presence of cardiac failure and as
parameter of therapeutic effect. In Becker and Duchenne muscular dystrophies (BMD
and DMD) dilated cardiomyopathy (DCM) with cardiac failure is frequently
observed among patients and carriers. Although the curvilinear relationship
between BNP and systolic dysfunction is known, the relationship between the NT-proBNP
level and the presence of DCM in patients and carriers is not. To assess whether
NT-Pro BNP can serve as predictor of dilated cardiomyopathy in DMD/BMD patients
and carriers, we included 394 individuals (84 DMD (17%), 92 BMD (24%), 147 DMD-carriers
(37%), and 81 BMD-carriers (21%)). All were interviewed, neurologically examined
and the following tests were done. NT-proBNP in peripheral blood was measured
(normal: <0.35 pmol/l). M-mode and 2D echocardiography was used to asses the
presence of DCM, defined as an enlarged left ventricle corrected for age and
height with a global left ventricle dysfunction or fractional shortening less
than 28%. A χ2
test was used for associations and ROC-analysis for sensitivity and specificity
of NT-proBNP for DCM. In DMD patients mean age was 13.6 years (SD 6.8), 62% was
wheelchair bound. The mean age of BMD patients was 30 years (SD 13), 73% had
some functional weakness of the leg muscles. Of the DMD-carriers (mean age 44
years, SD 11.8) and of BMD-carriers (43.3 years, SD 12.4), 14% and 13%,
respectively, had myalgia, cramps or weakness. Overall, clinical symptoms of
cardiac failure were present in 39%. The mean NT-proBNP was 0.64 pmol/l (SD
0.64). 62 (17%) out of 365 individuals in whom echocardiography was feasible,
had DCM. Nine patients with DCM had a normal NT-proBNP, none were treated for
DCM. The association between an elevated NT-proBNP and DCM was not significant (p = 0.12).
A ROC-analysis showed no NT-proBNP value where sensitivity and specificity of
any significance was reached (area under ROC curve = 0,58). NT-proBNP is not
associated with echocardiographic defined dilated cardiomyopathy in DMD/BMD
patients and carriers.
8) Revising the cardiac phenotype of Duchenne muscular dystrophy
L.W. Markham, A. Barone, K.
Kinnett, R. Spicer, B. Wong,
D.W. Benson and L. Cripe
- USA
Duchenne muscular dystrophy (DMD) is a progressive skeletal and cardiac myopathy
with a reported classic electrocardiogram (ECG) consisting of sinus tachycardia
along with additional ECG abnormalities. Inappropriate sinus tachycardia has
been postulated as precipitating the development of ventricular dysfunction. The
objectives of this study were to determine the prevalence of sinus tachycardia
and other ECG abnormalities in association with ventricular dysfunction. We
hypothesized that the DMD ECG profile would change with age and the degree of
ventricular dysfunction. From 1991 to 2006, 105 DMD boys had 503 ECGs and 322
echocardiograms performed. Subjects were divided into five groups based on age
of initial cardiac evaluation – A, 0–5 years (n = 17); B, 6–10 years (n = 34);
C, 11–15 years (n = 24); D, 16–20 years (n = 19); and E, >20 years
(n = 11). Left ventricular (LV) dysfunction was defined as a shortening
fraction <28%. By group, mean age in years at evaluation was: A, 4.3; B, 8.5; C,
13.5; D, 18.5; and E, 23.1. Sinus tachycardia (A, 0%; B, 12%; C, 30%; D, 48%;
and E, 55%) increased significantly with age (p < 0.01). The percent with
LV dysfunction (A, 5%; B, 15%; C, 33%; D, 53%; and E, 64%) also increased
significantly with age (p < 0.01). Sinus tachycardia occurred in
association with LV dysfunction (p < 0.001). Sinus tachycardia has a
sensitivity of 84%, specificity of 95%, predictive value positive of 87% and
predictive value negative of 93% for LV dysfunction. Additional ECG
abnormalities were frequently noted in later stages of cardiac disease. In DMD,
sinus tachycardia increases with age, rarely occurs prior to LV dysfunction, and
is temporally related to LV dysfunction. The “classic” ECG findings of DMD as
reported in the literature are only seen late in the cardiac disease process.
Additional ECG abnormalities become more prevalent with age and correlate with
the degree of ventricular dysfunction
9) Impaired platelet adhesion on collagen surfaces and secretion defect in Duchenne muscular dystrophy patients
V. Labarque,
N. Goemans,
K. Freson, M.F. Hoylaerts and C. Van Geet
- Belgium
1Center for Molecular and Vascular Biology, Leuven, Belgium
2University Hospital Gasthuisberg, Leuven, Belgium
Duchenne muscular dystrophy (DMD) patients experience a significantly increased
blood loss during spinal surgery without spontaneous bleeding tendency. Previous
studies explained this by decreased platelet adhesion to glass beads, due to a
deficiency of glycoprotein IV [Forst, Neuromuscul Disord 1998] or by the absence
of an isoform of dystrophin, Dp71, in platelets [Austin, J Biol Chem 2002].
Others hypothesized the defect of primary hemostasis in DMD could be due to
impaired vessel reactivity [Turturro, Neuromuscul Disord 2005]. However, none of
these hypotheses have been confirmed unequivocally. In this study, we further
investigated platelet function in DMD patients. We studied 11 patients in whom
the diagnosis of DMD was confirmed on muscle biopsy or by mutation analysis.
They showed no coagulation abnormalities and had a normal von Willebrand factor
antigen and function as well as a normal expression of GP Ib, IIb and IV.
Ristocetin- and ADP-induced platelet aggregations were decreased in 36% and 18%
of the patients, respectively. During perfusion studies with whole blood,
platelet adhesion on calf skin collagen was reduced at 2700/s (P = 0.01)
as well as at 1300/s (P = 0,048). Patients with absent Dp71 (n = 2)
did not show a reduced platelet adhesion. However, one of our patients showed a
decreased ATP-secretion on stimulation with ADP or collagen, three others had an
impaired serotonin release. Platelet Function Analysis, shape change,
FAK-phosphorylation and spreading experiments on fibrinogen were normal. In this
study, we found that DMD patients manifest an impaired platelet adhesion on
collagen. However, we could not confirm a role for GP IV or Dp71, while at least
in some patients, this platelet dysfunction could be explained by a secretion
defect. As the platelet dysfunctions are minor and variable, we conclude that
the increased blood loss in DMD patients could be due to the concurrence of
reduced platelet function and disturbances in local factors, such as vessel
reactivity and endothelial function. Future studies should focus both on these
environmental factors as well as on defects in pathways following platelet
activation.
10) The therapeutic effect of myostatin-blockade on muscular dystrophic mice and gene expression analysis of the treated muscles
S. Noguchi, M. Fujita,
T. Sasaoka and I. Nishino - Japan
Several therapeutic studies, such as gene therapies, cell implantations and
pharmaceutical therapies, have been attempted toward muscular dystrophies.
Recently, two growth factor-related molecules were highlighted by showing the
effects on improvement of dystrophic phenotype of mdx mice. One was insulin-like
growth factor-1 (IGF-1), and another was myostatin. Previously, we reported the
therapeutic effect of IGF-1 on sarcoglycanopathic mice. In this study, we made
recombinant folistatin-related gene (FLRG) protein in cultured cells and
measured its blockade activity against myostatin in vitro. Then we administered
recombinant FLRG protein into sarcoglycanopathic mice (sgcb−/− mouse at 3–5
weeks) and tried to evaluate its effect on their dystrophic phenotypes by
pathological observation on comparison with those of IGF-1 administration. After
administration of recombinant FLRG by osmotic pump implanted subcutaneously for
4 weeks, serum creatine kinase level was significantly decreased after
treatment. In pathological examination, the gastrocnemius muscles showed
remarkable reduction in necrotic fibers and increase in centrally nucleated
fibers similarly to IGF-1 administration. Using oligonucleotide arrays with
22,000 gene probes, we also analyzed expression profiles of skeletal muscles of
FLRG-administrated sgcb−/− mice. FLRG has a therapeutic effect on muscular
dystrophic mice in similar levels as with IGF-1. Microarray analysis is a
powerful tool for evaluation of the therapeutic effect.
11) The effects of prednisone on exercised-induced muscle damage in mdx mice
J. Collins,
B. Wong,
A.
McCullough
and J. Quinlan
- USA
Dystrophin deficiency results in muscle membrane instability and exercise
enhances contraction-induced injuries in dystrophin-deficient muscles.
Corticosteroids are known to delay progression of muscle weakness in children
with Duchenne Muscular Dystrophy, but the mechanism of action is unclear. To
evaluate the effects that prednisone had on exercised-induced muscle damage in
mdx mice. mdx mice were treated with prednisone or placebo for 4 weeks. They
were subjected to either a one time exhaustive run or a 4 week gentle daily run
on a rate-controlled treadmill. Intra-peritoneal Evans blue dye (EBD) was
injected thirty minutes prior to the exhaustive or final daily run.
Histopathological markers of membrane stability (EBD penetrated fibers) and
necrosis (CD11b) were examined in mdx rectus femoris muscles. In mice with
exhaustive runs, the mean EBD positive area was 6.2 ± 1.6% (SEM) in
prednisone-treated mice (n = 6) compared to 12.6 ± 2.5% (SEM) in
placebo-treated mice (n = 6, p < 0.05). No significant difference
was seen in the prednisone (n = 5, 6.4 ± 1.5%) and placebo (n = 5,
8.8 ± 1.4%, [p < 0.06]) groups with daily exercise. The mean CD11b
stained areas were significantly different in prednisone-treated (6.9 ± 0.65%,
n = 5) daily run mice compared to placebo (13 ± 2.9%, [n = 5, p < 0.05]).
Prednisone stabilized the mdx sarcolemma in strenuous exercise and alleviated
muscle cell damage and necrosis.
12) Quantitative ultrasound measurements of bone density DMD and SMA patients
A. Berardinelli, K. Gorni, L.
Pedrotti, R. Mora, B.
Bertani and E. Fazzi
- Italy
Osteoporosis is characterized by loss of both bone mass and microarchitectural
integrity, resulting in an increased risk of fractures with associated morbidity
and mortality. Bone mass in the elderly is related to the rate of involutional
bone loss and mainly to the peak bone mass reached in childhood and adolescence.
The peak bone mass appears as being the key issue problem in prophylactic
strategy of osteoporotic therapy. Reduced bone density is now well recognized in
children and adolescents affected by Duchenne Muscular Dystrophy (DMD) even
before motor impairment is clinically relevant; only one study has been
performed so far about Spinal Muscular Atrophy (SMA) patients as far as we know,
surprisingly a normal bone density nevertheless an even more severe motor
impairment. The aim of this study was to determine quantitative ultrasound
velocity, which is related to bone mass, in patients with DMD and SMA. The work
is in progress and more patients will be included by next summer. Each patient
is included accordingly with his follow-up time table. The sample so far
consists of 14 patients, 12 males and 2 females; 4 non-ambulant; 10 affected by
Duchenne (DMD), 2 by Becker Muscular dystrophy (BMD) and 2 by Spinal Muscular
Atrophy (SMA). The rage range was from 3 yrs 8 mths to 18 yrs. Quantitative
ultrasound velocity was measured by Sunlight Omnisense, a device designed to
measure SOS (Speed of Sound) at multiple skeletal sites, by axial transmission
method. The purpose of the study is to investigate whether osteopenia/osteoporosis
is always associated with neuromuscular pathology and if that is related to
motor impairment. In order to critically interpret the sonographic results of
the children included in our study, we have drawn a diagram of reference with
the data obtained by the same device in the control group. Speed of sound
values, indicative of bone density, obtained in children affected by DMD and SMA
were compared with those obtained by means of the same device in a control group
of 700 Italian healthy children and adolescents aged 6–19 years. Correlation
between degree of disability and SOS has been analysed. 5 subjects had normal
SOS values, 6 were classified as osteopenic and 3 as osteoporotic. Only 1 child
in the “osteopenic” group had a possible pathologic bone fracture in the cuboid
some years ago. Six DMD patients are on steroids. Due to the small sample we
cannot perform any statistical analysis so far. As described in the literature
the most of our DMD patients had reduced SOS, but we also found some DMD in the
“Normal” group, apparently with no correlation with steroid therapy, age, or
motor impairment. Both of the younger DMD patients (3 years 8 months/3 yrs 11
mths) are in the “not normal” group.
13) Bone health in Duchenne muscular dystrophy
B.L. Wong,
M.Y. Shao, M. Hurtubise, A. El-Bohy, M. Knue,
P. Morehart
and J.E. Heubi - USA
Patients with Duchenne muscular dystrophy (DMD) are at risk for osteoporosis and
fractures secondary to muscle weakness, loss of mobility and chronic
corticosteroid therapy. To assess the prevalence of reduced bone mineral density
(BMD) and fractures in patients with DMD, a cross-sectional study of 45 patients
with DMD was conducted between June and August 2005. The primary outcome
variable was BMD of distal femur [DF]] (R1 – cancellous bone, R3 – cortical
bone, R2 – between R1 and R3) and lumbar spine (LS) as measured by DEXA z
scores. The secondary outcome variables were fractures (per history and
radiographs of spine and lone bones), DEXA lean body mass adjusted for weight,
cumulative corticosteroid dose, calcium intake, serum 25 OH vitamin D levels and
functional mobility level. Patients: n = 45 (ages 3.3–26, mean age 12.3
years), 24 on chronic steroids, 22 wheel chair dependent and 23 independent
ambulators. For all DMD patients, DEXA z score (±SEM) for LS was −1.05
(±0.20); DF R1: −2.9 (±0.32), R2: −2.56 (±0.43), R3: −2.60 (±0.48) and whole
body (WB): −1.78 (±0.25). DEXA z scores for LS were −1.08 (±0.34) vs
−1.03 (±0.25); DF R1: −4.36 (±0.45) vs −1.63 (±0.23) [p < 0.0001]; R2:
−4.33 (±0.63) vs −1.02 (±0.37) [p < 0.0001]; R3: −4.71 (±0.69) vs −0.76
(±0.38) [p < 0.0001] for non-steroid treated vs. treated subjects,
respectively. LS, all 3 DF and WB DEXA z scores did not correlate with 25
OH Vitamin D levels and dietary calcium intake. DF R1, R2 and R3 z scores
correlated with weight adjusted lean body mass (p < .0001) and with
functional mobility levels (p < .0001). Analysis of covariance for
comparing the steroid group and the non-steroid group adjusted for one of the
four covariates (BMI, height, calcium intake and functional mobility level)
found height and functional mobility level separately had significant effects as
a covariate for the 3 DF z scores (p < 0.0001). When all above
parameters were placed in the regression model as covariates with steroids,
functional mobility level had significant effects for all 3 DF scores (p < 0.005),
and height was significant for DF R3 scores (p = 0.04). Calcium intake
was 1404.1 (±387.7) mg/day. Vitamin D intake was 498 (±62) IU/day and 25 OH
vitamin D level was 26.78 (±1.67) ng/ml. Twenty of 45 (44%) patients had a
history of fractures; 9/20 (45%) were on steroids; 5 patients had spine
compression fractures (all on steroids). WB DEXA z scores correlated with
fracture history (p = 0.0003). BMD is reduced in patients with DMD
despite adequate calcium and vitamin D intake. Osteoporosis in the lower
extremities is more profound in non-steroid treated DMD patients compared to
steroid treated patients and is correlated with worsening functional mobility
and decreased muscle mass. Further larger studies are needed to define the risk
factors for osteoporosis and fractures in DMD patients to enable effective
treatment interventions.
14) Functional ability monitoring in Duchenne muscular dystrophy using posture and walking time recording in a home environment
C. Bloetzer,
B. Najafi, K. Aminian and P.Y. Jeannet
-
Switzerland
Developing new tools to better assess disease progression in neuromuscular
disorders is important, particularly in the light of new therapeutic
possibilities. In this pilot study, the daily functional ability of Duchenne (DMD)
patients and healthy controls was measured using a small and light, wireless
ambulatory system (ASUR) attached to the child’s t-shirt. ASUR is composed of
three accelerometers and one gyroscope which identify body postures and periods
of walking. Seven DMD patients (age 4–10 years) and 2 healthy controls (age 5
and 7 years) were studied in their home environment for one day while parents
kept a diary of their activities. A validated motor function measure scale (MFM)
was performed in patients 6 years or older (n = 5) before the recordings.
There was good correlation of recorded data with parent’s diary. For patients 8
years or younger (n = 5), mean time spend sitting (Si) was 31% ± 6.5;
standing (St) 41% ± 6.4; lying (Ly) 3% ± 1.7; walking (Wa) 25% ± 4.5; mean
number of walking episodes (WE) was 371 ± 165; and mean number of continuous
walking periods for more than 30 s (Wp30) was 54 ± 17; mean MFM score was
84.4/100 (n = 3). In patients older than 8 years (n = 2), Si was
53.5% ± 6; St 28% ± 13; Ly 5.5% ± 8; Wa 13% ± 1; WE 302 ± 143; Wp30 26.5 ± 5;
mean MFM score 87/100. In controls, Si was 16% ± 4; St 54% ± 10; Ly 9% ± 8; Wa
21% ± 3; WE 567 ± 83; Wp30 29 ± 1. One patient (age 4 years) monitored before
prednisolone treatment and one month later increased Wa from 22 to 30%, We from
650 to 859, Wp30 from 46 to 93. Older and more disabled DMD patients spend more
time sitting, less time standing and walking and had less walking episodes than
younger patients and controls. Improvement of functional ability after
initiation of steroid treatment was quantified in one patient. These promising
preliminary results on a small cohort need to be confirmed by a larger study,
currently under way.
15) Is there a relationship between hamstring length and function in ambulant boys with Duchenne muscular dystrophy?
C. Nicholson, M. Main, E.
Mercuri and F. Muntoni - UK and Italy
Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder
characterised by weakness, falls, toe walking, joint contractures, difficulty
rising from the floor and early loss of independent ambulation. It has been
reported that tendo-achilles (TA) tightness interferes with function in ambulant
boys with DMD and physiotherapy treatment including stretches and use of night
splints, helps to maintain TA length. No studies found have considered the
effect of hamstring length on function in ambulant boys with DMD, although it is
usual for physiotherapists to teach hamstring stretches as part of daily
management. Popliteal angle has been found to be a reliable measure of hamstring
length with studies in normal paediatric populations showing increasing
tightness up to the age of 9 years. These studies give normal range as being
20–50°. Our aim was to determine whether there was a significant relationship
between hamstring length and function and try to establish when hamstring
stretches are indicated for boys with DMD. Sixty boys with a confirmed diagnosis
of DMD seen regularly at Hammersmith Hospital were included in the study.
Measurements of hamstring length, TA and ilio-tibial band (ITB) tightness, timed
rise from the floor, Hammersmith motor ability score, and muscle power measured
using the Medical Research Council (MRC) scale, were recorded during routine
assessments. The results show, of the 60 boys, only 6 had a popliteal angle
greater than 50°. Using Spearman’s correlation coefficient there was no
significant relationship between hamstring length and functional score (p < 0.503)
with a weak correlation between hamstring length and timed rise from the floor (p < 0.006).
There was a significant relationship between function and hip extensor power (p = 0.001)
and between timed rise from the floor and hip extensor power (p < 0.001).
Hamstring length, therefore, was not proven to be a major factor in causing
functional difficulties in ambulant boys with DMD. Further, from our results,
hamstring tightness does not appear to be a common feature of ambulant boys with
DMD and the appropriateness of routine hamstring stretches for these boys, has
not been established.
16) Predictive factors for the development of scoliosis in Duchenne muscular dystrophy
M. Kinali, R.K. Knight,
M. Main, E. Mercuri, S.
Messina, A.Y. Manzur and F.
Muntoni - UK and Italy
Scoliosis is a complication in up to 90% of boys with Duchenne muscular
dystrophy (DMD). Physiotherapy, rehabilitation in lightweight knee–ankle–foot
orthoses (KAFOs), and glucocorticosteroids contribute to prolongation of walking
and standing and might reduce the risk of scoliosis development. We evaluated
possible predictive factors for development and progression of scoliosis in a
large DMD population. Case notes of 123 boys with DMD
> 17 years,
followed at our centre over a 10 year period were reviewed. Stepwise multiple
regression analysis was used to correlate the age of onset and severity of
scoliosis with: glucocorticosteroid treatment, ages at loss of ambulation and
rehabilitation into KAFOs, length of KAFO usage, lower limb contracture
asymmetry and age at loss of standing. 95/123 boys (70%) developed scoliosis
with a Cobb angle 30°.
Mean age (SD) at scoliosis onset was 12 years 8 months (SD 1 year 7 month).
37/123 boys (30%) received intermittent prednisolone (0.75 mg/kg/day, 10
days/month) for a median 1 year (range 2 month to 9 year), starting between
7 years 8 months and 12 years 5 months (mean 9 year 7 month). 96/123 (78%) were
rehabilitated into KAFOs at 10 years 2 months (SD 1 year 7 month). Ages at loss
of ambulation in KAFOs were 12 years 2 months and at loss of standing 12 years
10 months (SD 2 year 1 month). Forty-three boys (35%) had scoliosis surgery at
15 years (SD 1 year 2 month; preoperative Cobb angle 62.2° [SD 15.9°]). Of the
80/123 boys managed conservatively, including those who refused surgery, 12/80
had no scoliosis, 16/80 had mild scoliosis (31–120°). Age at onset of scoliosis
correlated with loss of independent ambulation (p < 0.005). Combination
of age at rehabilitation into KAFOs and duration of glucocorticosteroid
treatment correlated with scoliosis severity at 17 years (p < 0.005). Our
data demonstrate that age at loss of independent ambulation is an important
predictive factor for scoliosis development. Other aspects of intervention such
as rehabilitation into KAFOs and glucocorticosteroid treatment may also
influence scoliosis outcome.
17) Does deflazacort treatment impact the surgical outcomes for boys with Duchenne muscular dystrophy?
W.D. Biggar, V.A. Harris and
B.A. Alman - Canada
The objective of this study was to compare the surgical outcomes for 3 groups of
boys with DMD: (a) 41 steroid-naïve boys, (b) 8 boys previously treated with
deflazacort, and (c) 9 boys on deflazacort at the time of surgery. This was a
retrospective review of 58 boys with DMD between 1990 and 2006 and followed at
Bloorview Kids Rehab. For the 41 steroid-naïve boys who had spine stabilization
surgery at age 15.1 ± 1.8 years, 39 of 41 required assistance for feeding within
1 year of surgery, 13 had severe weight loss, 9 had severe leg/hip pain lasting
longer than 12 weeks, 6 had an infection at the surgical site and 4 of those 6
required a surgical revision. Eight boys had been treated previously with
deflazacort: (age deflazacort started 8.6 ± 2.4 years, age deflazacort stopped
12.4 ± 2.9 years, years on deflazacort 3.8 ± 1.4, age at surgery 14.9 ± 2.2
years). None had deflazacort within 1 year of surgery (2.5 ± 1.7 years).
Pneumonia developed in 1 boy, a surgical infection in 3 boys, and 1 of these 3
developed Clostridium difficile colitis. None had severe weight loss.
Nine boys were on deflazacort (0.2–0.7 mg/kg/day) at the time of surgery: (deflazacort
started 11.5 ± 1.6 years, years on deflazacort 3.2 ± 1.3, age of surgery
14.7 ± 2.2 years). One boy had severe post-operative leg/hip pain. None had
infections, poor wound healing or severe weight loss. All remained independent
for feeding more than 1 year after surgery. In addition,we reviewed the surgical
outcomes for another 135 boys ages 4–21 years treated with daily deflazacort, 2
had a typical presentation for acute appendicitis and a normal recovery, 3 had
fractured femurs treated uneventfully with internal fixation, and 10 had one or
more elective dental extractions. Our findings suggest that surgical outcomes
are not adversely affected by deflazacort treatment in boys with DMD.
Furthermore, in this small series, when deflazacort is started in older boys and
at a low dose or if deflazacort is started in younger boys and then
discontinued, the progression of scoliosis seems unaffected.
18) CINRG pilot trial of oxatomide in steroid-naive Duchenne muscular dystrophy
G.M. Buyse, D.M. Escolar, N. Goemans, E.
Henricson, M. van den Hauwe, A. Jara Vallejos, C. Shao,
K.M. Patel, R. McCarter,
R. Leshner, J. Florence
and J. Mayheww - Belgium and USA
Mast cells have been implicated in the pathophysiology of Duchenne muscular
dystrophy (DMD) and its animal models. In addition the mast cell stabilizer
oxatomide has shown promise in a pre-clinical screening platform that uses the
homologous mdx mouse model of DMD. With CINRG (Cooperative International
Neuromuscular Research Group), we have tested the hypothesis that oxatomide
specifically targets the pathophysiological cascade of DMD by blocking the mast
cell mediator cascade and inhibiting dendritic cell’s release of cytokines, and
hence may improve muscle strength or delay progression of muscle weakness in DMD
patients. We tested the efficacy and safety of oxatomide in 14 ambulant
steroid-naive DMD boys aged 5–10 years, in a pilot open-label two-center
clinical trial with a 3 months medication-free lead-in period followed by a 6
months treatment period with oral oxatomide (30 mg/day). Drug efficacy was
tested by measuring muscle strength quantitatively (QMT, 10 muscle groups) and
manually (MMT, 34 muscle groups), and by timed functional tests. Repetitive
measure analysis was applied using linear mixed-effects models. Comparison of
linear evolutions of muscle strength and function between lead-in and treatment
periods showed no significant differences. There was a strong trend that
patients treated for 6 months with oxatomide showed less deterioration in QMT
measured muscle strength than in an historical matched control group. There were
no serious adverse events and overall oxatomide was well tolerated. Within the
limitations of a smaller non-controlled trial, we conclude that 6 months
oxatomide treatment does not improve muscle strength or function in a group of
5–10 year old DMD patients. A minor disease-modifying effect of oxatomide in DMD
cannot be excluded.
19) Lower urinary tract symptoms in patients with Duchenne muscular dystrophy
E. van Wijk, E.J. Messelink
and I.J.M. de Groot - Netherlands
Duchenne muscular dystrophy (DMD) is a progressive disease that mainly affects
striated muscles. However, involvement of gastrointestinal muscles (smooth
muscles) and bladder dysfunction have also been reported. Lower urinary tract
symptoms (LUTS) are frequently reported by boys with DMD at the outpatient
rehabilitation clinic, though the prevalence of LUTS and the effect of LUTS on
social activities in the DMD population are unknown. Therefore, we conducted a
questionnaire-based survey on the prevalence and social effects of LUTS in the
Dutch DMD population. A postal questionnaire was sent to members of the Dutch
DMD patient organisations. The questionnaire was based on International
Continence Society standards and contained general questions and specific
questions about LUTS. Boys and men with DMD from the age of four were included.
DMD was confirmed by DNA or muscle biopsy. Two hundred and one out of 217
respondents were included. 171/201 patients (85%) had one or more LUTS. One
hundred and twenty-nine of the 171 (67%) patients with LUTS had 3 or more
complaints. Post micturition dribble, straining, feeling of incomplete emptying,
slow stream and (urge) incontinence were most frequently mentioned. 44/201 (22%)
were too late toilet trained and 5 patients above the age of 12 were
incontinent. The effect of LUTS on social activities varied per complaint,
though a mean of 42% of patients (range 18–76%) experienced the LUTS as a
problem. In 51/201 (25%) patients, quality of life was reduced by LUTS. 47/171
(27%) of patients had never mentioned their complaints to a doctor. The LUTS
were not strongly correlated to age, functional status, scoliosis operation
and/or constipation. Virtually all boys and men with DMD have LUTS, and often
reduces quality of life. LUTS seem to be integrated, though underreported
symptoms of DMD, and are not related to progression of disease. Further research
is necessary to unravel the pathophysiology of LUTS in DMD and to direct future
treatment.
20) Depression and functional evaluation in Duchenne muscular dystrophy according to family functionality
R.E. Escobar, A. Miranda, M. Alfaro, C.K.
Rocha, F. Calderon and E. Espejel - Mexico and USA
Depression in Duchenne muscular dystrophy (DMD) could be the result of the
progression of natural history of this disorder, but some other factors could
also be related such as family functionality.
The aim of this study was to
compare depression and functional evaluation in DMD patients with family
functionality. A comparative study was conducted. Patients with DMD diagnosed
through clinical, enzymatic and electromyographic means were selected, aged
between 8 and 14 years. Family functionality was evaluated with a Familial
Functionality Profile questionnaire applied in parents and patients and
according to the results two groups were formed, patients with functional and
dysfunctional families. Depression was evaluated with the Children Depression
Scale questionnaire. Functional evaluation were assessed through Brooke, Vignos
and Swinyard functional grading scales. Family functionality and depression
questionnaires have been validated for Mexican families. Analysis was performed
with descriptive statistics, Mann-Whitney U test and chi squared or Fisher exact
test. Thirty-five patients were invited but only 21 accepted to participate.
There were no significant statistical differences between the groups in
depression or functional grading scales (p > 0.05). But there was a
slight tendency to increase depression values according to questionnaire results
and a decrease in functional grading scales. In this study, we found that there
is no differences between depression and functional evaluation in patients with
Duchenne muscular dystrophy according to family functionality. It is possible
that the 14 patients who refused to participate maybe were too depressed to do
it.
21) Protease inhibitors as potential treatment strategy for muscular dystrophies
T. Meier
- Switzerland
Protease inhibitors as potential treatment strategy for muscular dystrophies
Thomas Meier Santhera Pharmaceuticals, Liestal, Switzerland;
www.santhera.com.
Absence of the functional dystrophin protein in Duchenne muscular dystrophy (DMD)
leads to elevated levels of intracellular calcium which is considered as a key
step in the cellular pathology of the disease. The ubiquitous calcium-dependent
cytosolic cysteine protease calpain is activated in dystrophin-deficient muscle,
and consequently, calpain inhibition is regarded as a potential treatment option
for DMD. Subsequent proteolysis of myofibrillar proteins by the ubiquitin–proteasome
pathway further contributes to the loss of muscle tissue. Genetic and
pharmacological studies validate both calpain and the proteasome as potential
drug targets for DMD. Since currently available calpain and proteasome
inhibitors are not suitable for the chronic use in DMD patients we have
developed and optimized novel calpain/proteasome inhibitors as a potential
treatment option for DMD. These dual-property inhibitors have superior overall
pharmacological properties as compared to reference calpain and proteasome
inhibitors. We further demonstrate that a selected calpain/proteasome inhibitor
accumulates dose-proportionally in muscle tissue where it significantly improves
quantitative histological parameters, restores the expression of beta-dystroglycan
at the sarcolemma and ameliorates motor performance of mdx mice. Our approach to
simultaneously inhibit calpain and the proteasome not only qualifies as
promising pharmacological treatment option for DMD but for all forms of muscular
dystrophies where impaired calcium homeostasis and elevated proteolytic activity
contributes to the manifestation of the disease.
22) Antisense-induced exon skipping in Duchenne muscular dystrophy patients
A. A.M. Janson, I.H.B. Ginjaar, A.
Aartsma-Rus, G.-J.B. van Ommen, A. van der Kooi,
S. de Kimpe, P. Ekhart, G. Platenburg, J.J.G.M. Verschuuren and J.C.T. van
Deutekom - Netherlands
Antisense oligonucleotides (AONs) are emerging as small molecule drugs with a
high, sequence-specific, corrective potential on RNA level. Especially for
Duchenne muscular dystrophy (DMD), AONs have demonstrated to be an efficient and
relatively simple and safe alternative to gene therapy approaches based on gene
replacement. By inducing specific exon skipping, AONs allow restoration of the
open reading frame of mutated DMD transcripts and turn on the synthesis of
internally truncated, but largely functional Becker-like dystrophins. We have
successfully demonstrated its high efficiency and specificity in cultured muscle
cells from a series of DMD patients affected by different mutations, as well as
in muscle tissue of the mdx and hDMD mouse models. Over the last 6 months, we
have performed a small-scale, single-centre, clinical trial study at the Leiden
University Medical Centre. It was based on a single, local, intramuscular
injection of an antisense oligo (with a 2′OMePS RNA chemical backbone) that
efficiently induces the skipping of exon 51. Six Dutch DMD patients were
selected in the age group of 8–16 years, with well-characterized mutations
correctable by exon 51 skipping (i.e., deletion of exons 49–50, 50, or 52). They
were pre-screened by confirming the mutation on RNA level and by showing the
therapeutic effect of the antisense oligo in cultured muscle cells. The oligo
was administered in a 1–2 cm3 area in the tibialis anterior muscle in
which the progression of muscle damage was determined to be limited by MRI
analysis. A small biopsy of the treated area was taken at 4 weeks
post-injection. Samples were analyzed for local exon 51 skipping and novel
dystrophin production. In addition, patients were carefully screened for any
adverse effects.
23) Rescue of dystrophin in the GRMD dog by multi-exon skipping using engineered U7 snRNAs
A. Vulin, A. Goyenvalle,
I. Barthélémy, F. Leturcq,
J.C. Kaplan, J. Chelly,
O. Danos, S. Blot
and L. Garcia - France
Among well described animal models of Duchenne muscular dystrophy, the GRMD dog
is likely the best model of DMD patients in terms of size and pathological
expression of the disease. GRMD is caused by a 3′ splice-site point mutation in
intron 6, which induces the skipping of exon 7 and thus results in a frame shift
that prematurely aborts dystrophin synthesis. By forced exclusion of two exons
(6 and 8), it is possible to restore an open-reading frame. Antisense
oligonucleotides (AO) allow specifically to target and inhibit individual genes
for the treatment of the disorder. However, since the AO are not self-renewed,
they cannot achieve long term correction. To overcome this limitation, we have
introduced antisense sequences into small nuclear RNAs (snRNA) and vectorized
them in AAV vectors. To determine efficient AO, we tested in particular exon
splicing enhancer (ESE) motifs, which target internal exon sequences, crucial to
pre-mRNA splicing. We designed different 2′O-methyl oligoribonucleotides
which were tested first in vitro. Then we have designed AAV vector harboring
chimeric U7 snRNA carrying efficient antisense sequences against exons 6 and 8
of the dog dystrophin gene, which were injected in GRMD muscles. Among different
antisense oligonucleotides tested in GRMD, the combination of 2 AONs targeting,
respectively, ESE of exon 6 and 8 induced delta5–9 in-frame skipping. In this
study, we show the efficacy of these ESE sequences for exon-skipping in culture
myotubes and in vivo. We also detected an almost complete restoration of
dystrophin expression after intramuscular injections and systemic delivery of
AAV-[U7-ESE6/U7-ESE8] vectors in GRMD. In this study, we provide evidence that
efficient skipping of two exons can be achieved in GRMD through U7snRNA
shuttles. Theoretically over 75% of Duchenne patients could benefit from
skipping of a single exon and multi-exon skipping would significantly extend
this percentage to most DMD mutations
24) Vascular endothelial growth factor gene transfer using adeno-associated viral vectors stimulates skeletal muscle regeneration and enhances muscle function in mdx mice
S. Messina, M. Aguennouz, A. Bitto,
A. Migliorato, M. Giacca,
F. Squadrito and G. Vita - Italy
Vascular endothelial growth factor (VEGF) is a major regulator of blood vessel
formation during development and in the adult organism. Several evidences
support its role in myogenesis and in myoblast migration and survival. Recently,
it has been reported that the delivery of VEGF using adeno-associated-virus (AAV)
vectors reduces muscle damage and promotes muscle regeneration in experimental
models of muscle necrosis in mouse. We tested whether this effect was
reproducible in mdx mice evaluating and we also measured the effect on
muscle function by using a grip meter, CK level and morphological and
biochemical parameters. Ten 4-week old mdx and ten wild-type mice were
treated with intramuscular administration of AAV-VEGF (3 × 108 viral particles)
or AAV-LacZ (3 × 1011 viral particles) into the biceps and tibialis anterior
(TA) muscles. Evaluations were performed one month after injection. VEGF-AAV
treatment increased the expression of VEGF. Mdx mice treated with
AAV-VEGF showed a significantly higher forelimb strength than AAV-LacZ controls
(+19,5%, p < 0.05) as well as higher strength normalised to weight
(+14,9%, p < 0.05). There were no differences in CK levels between VEGF-
and LacZ-treated mdx mice. Quantitative morphological evaluation of VEGF-treated
biceps and TA muscles showed a reduction of necrotic area (p < 0.05) and
an increase of regenerating area (p < 0.05). We report herein the novel
observation of a pro-regenerative role of VEGF in mdx mice and of its
beneficial effects on functional parameters. Further studies are needed to
better clarify the mechanisms underlying the VEGF-induced benefit in mdx mice
and to investigate possible therapeutic implications in Duchenne muscular
dystrophy.