Abstracts that will be presented in Annual Meeting of American Academy of Neurology, Seattle, April-25-May-2, 2009
[P06.105] Reduction of TGF Beta 1
Using an Oral TNF Alpha Inhibitor in mdx Dystrophic Mice
Leonardo Horacio Brito, Lucas Scatino Saad, Vinicius Oliveira,
Eduardo Augusto Correa Barros, Rosangela Aparecida Dos Santos, Lucas Guimaraes
Ferreira, Maria Helena Catelli Carvalho, Maria Tereza Nunes, Szulim Ber Zyngier,
Alzira Alves Siqueira Carvalho, DAVID FEDER, Sao Paulo, Brazil
OBJECTIVE: To evaluate the effect of Bkt-104, an oral inhibitor of TNF-
in mdx mouse BACKGROUND: Tumor necrosis factor (TNF)-
is elevated in inflammatory myopathies as well in DMD. The precise role of TNF-
is unknown, and it is possible involved in the pathogenesis of fibrosis in
dystrophic muscle. Blunting TNF pharmacologically may be useful in preventing
muscle fibrosis in dystrophic muscles. DESIGN/METHODS: Long term effects
of oral BKT-104 was analysed on mdx mice, male, 8 weeks old; five mice received
BKT-104 30mcg/Kg 3 times a week and six received saline (double blinded,
placebo). Mice were submitted to exercise in ergometer 5 days/week, at a speed
of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12 weeks.
After 12 weeks, all mdx mice undergone to muscle biopsy and right quadriceps and
gastrocnemius were collected and stained with acid phosfatase and alkaline for
histological evaluation. Left gastrocnemius was dissected, frozen in liquid
nitrogen, and stored at -80 C. The tissue was used for Real-time PCR and mRNA of
cytocines were measured. RESULTS: The results of this study didn't show
any improvement on muscular strength or histological alterations in BKT-104
tretment of mdx mice. There were no difference in genic expression of myostatin
but a significant reduction of TNF-
and TGF beta 1 in treated mice. CONCLUSIONS/RELEVANCE: Presently, TNF
inhibitors are only available as an injectable or infusion. BKT104 is a small
molecule (sulfated disaccharide), an oral inhibitor of TNF-
(and consequential IL-8 production). BKT104 have show to be highly effective in
modulating inflammatory disease in animal models, especially those regulated by
TNF-.
This is a pilot study with the drug Bkt-104 in muscular dystrophy. The observed
reduction in TNF-
and TGF beta 1 demonstrate the potential of the drug in reducing the fibrosis
seen in muscular dystrophy. Acknowledge: Dr. Orly Eizenberg from Biokine
Therapeutics
[P06.088] Erythropoietin
Application in mdx Mice
A Pilot Trial
Alzira Carvalho, Michelle Remiao Ugollini, Augusto Cesar Santomauro,
Jr., Leticia Zaccaria Prates Oliveira, Victor Prates Pimentel Lioi, Rosangela
Aparecida Dos Santos, Lucas Guimaraes Ferreira, Maria Tereza Nunes, Maria Helena
Catteli Carvalho, Szulim Ber Zyngier, DAVID FEDER, Sao Paulo, Brazil
OBJECTIVE: To evaluate the effect of Erythropoietin application in an
experimental model of dystrophy, the mdx mouse BACKGROUND: Apart from its
erythropoietin function, erythropoietin (Epo) exerts also non-erythroid but
tissue protective functions. The potential physiological role of the
erythropoietin receptor (Epo-R) in skeletal muscle seems to be the control of
proliferation and differentiation of myoblasts and satellites cells. Also, it
has been suggested an important role of Epo in response to injury. DESIGN/METHODS:
The long term effects of intraperitoneal(IP) Epo injection on mdx mice, male,
were analysed. Seven mdx mice received Epo (1000 IU/kg/IP) and six received
saline. All animals were submitted to exercise in ergometer 5 days/week, at a
speed of 20 cm/s, 10 min/day. The muscle strength was measured weekly for 12
weeks. After 12 weeks of treatment, all mdx mice were undergone to muscle biopsy
and muscle samples were collected from right quadriceps and gastrocnemius and
stained for histological evaluation. Left gastrocnemius was dissected, frozen in
liquid nitrogen, and stored at -80 C. The tissue was used for Real-time PCR and
cytocines were measured. RESULTS: The results didm't show that 12 weeks
of Epo treatment was beneficial for improving muscular strength in mdx mice but
improved muscular strength during one week (week seven) in the treated group.
The histological analysis did not detect differences in necrosis and
regeneration between both groups. There were no difference in genic expression
of TNF alpha and TGF beta 1 but a significant reduction of myostatin in treated
mice. CONCLUSIONS/RELEVANCE: Myostatin reduction in erythropoietin
treatment in dystrophic mice could explain some effects of erythropoietin in
skeletal muscles. The lack of improvement in muscle strength for more than one
week may reflect the amount of Epo dose used or the period of treatment. A
higher dose and early use of Epo could be a potential application in our next
trial.
[P06.097] Behavioral Outcomes of a CINRG
Multi-Center, Double-Blind, Controlled Study Comparing High Weekly Versus Low
Daily Dose Prednisone Therapy in Boys with Duchenne Muscular Dystrophy
Yaron Fridman, Adrienne Arrieta, Fengming Hu, Lauren P. Hache, Jason
M. Weimer, Hoda Z. Abdel-Hamid, Diana Escolar, Avital Cnaan, Paula R. Clemens,
Washington, DC, Pittsburgh, PA
OBJECTIVE: To compare the behavioral effects of prednisone dosing
0.75mg/kg daily (daily) with dosing 10mg/kg weekly split over two consecutive
days (weekend) for Duchenne muscular dystrophy (DMD) using the Child Behavior
Checklist (CBCL). BACKGROUND: Prednisone prolongs ambulation of patients
with DMD,but may cause adverse behavioral effects that could be alleviated by
alternate dosing regimens. DESIGN/METHODS: We performed a secondary
analysis of the behavioral outcomes in a double-blind, multi-center,
placebo-controlled equivalence study of daily and weekend prednisone dosing. The
CBCL was administered at screening and months 1, 3, 6, 9, and 12. Group
comparisons were analyzed with paired t-tests, two-sample t-tests, and
statistical significance was defined at p=0.05. RESULTS: Fifty-six (28 in
each group) of 64 randomized participants completed the CBCL. The measures for
the majority of participants had CBCL scores in the subclinical range throughout
the study. Baseline comparison between groups showed slightly more problems in
externalizing (p=0.05) and aggressive (p=0.02) behaviors in the daily prednisone
group. Total problems at baseline were similar between treatment groups
(Weekend=52.18 sd=7.44, Daily=55.19 sd=11.00, p=0.24). Comparison of baseline to
one month showed no changes in behavior within weekend prednisone and an
improvement in total problems (p=0.029) and attention (p=0.017) with daily
prednisone. Following 12 months of treatment there were no significant
differences within the weekend group. There were significant decreases in total
problems, externalizing, attention, and aggressive behaviors (p<0.001, p=0.001,
p<0.001, p<0.001) with daily prednisone. There were also significant differences
between the daily and weekend groups (p=0.005, p=0.008, p=0.010, p=0.001),
respectively. CONCLUSIONS/RELEVANCE: This study demonstrated significant
improvement in total and externalizing behavioral measures determined by the
CBCL with daily prednisone dosing over 12 months, and no change in the weekend
group. Therefore, from a behavioral standpoint, daily dosing is preferred over
weekend dosing for those DMD patients having or at high risk of behavioral side
effects from prednisone therapy.
[P06.054] Expected Transaminase Levels in Muscular
Dystrophy with Chronically Elevated Creatine Kinase
Erika L. F. Hedderick, Carmen M. Coombs, Richard E. Thompson, Thomas
O. Crawford, Baltimore, MD
OBJECTIVE: To define the range of expected transaminase levels in
patients with chronically elevated creatine kinase levels due to muscular
dystrophy. BACKGROUND: Creatine kinase is prominently elevated in many
forms of muscular dystrophy, but serum transaminases ordinarily associated with
liver disease are elevated as well due to ongoing leakage of each from muscle.
Defining the relationship between serum levels of these enzymes is important to
both clinical trial design and clinical care in order to identify patients who
may have liver disease in addition to their muscle disorder. DESIGN/METHODS:
This retrospective review identified 195 patients with Duchenne, Becker, or limb
girdle forms of muscular dystrophy seen by the senior author between 1975 and
2008. All patients who had baseline laboratory measurements of serum CK, AST,
and/or ALT on the same day, in the absence of acute illness, were included.
RESULTS: Forty-seven patients had at least one assessment that met these
criteria. Nineteen of these patients had more than one set of laboratory tests
over the course over many years, yielding a total of 122 data sets for AST and
105 for ALT. There were 44 males and 3 females with the following diagnoses:
Duchenne (32), Becker (7), congenital muscular dystrophy (2), limb-girdle
muscular dystrophy (4) and manifesting carriers of dystrophinopathy (2). Median
age is 10.7, range 2.1 and 36.3 years. CK ranges between 41 and 29,595, and
naturally declines with age and loss of muscle mass. For AST, upper limit of the
95 percentile confidence interval for a given CK value is 199 + 0.022*CK. For
ALT, this value is 313 + 0.027*CK. CONCLUSIONS/RELEVANCE: These values
are important to identify potential treatment complications in pending clinical
trials.
[P06.089] Cell Therapy for Muscular Dystrophy:
CD34 Negative Muscle Derived Cells Present High Myogenic and No Adipogenic
Potential
Sabrina Sacconi, Didier Pisani, Noemi Clement, Claude Dechesne,
Christian Dani, Claude Desnuelle, Nice, France
OBJECTIVE: to identify, within myogenic muscle precursors, a cell
population with a high myogenic and low adipogenic potential in the perspective
of cell therapy for muscular dystrophy. BACKGROUND: Myoblast
transplantation in clinical trials is based on intramuscular injection of a
population of CD56 positive myogenic muscle precursors (MMP). This population
contains multipotent stem cells able to differentiate into distinct mesenchymal
lineages including adipocytes. Adipocyte accumulation is observed in human
dystrophic muscular diseases and stem cells transplanted in a muscle environment
permissive to fat development, may be committed towards adipogenesis at the
expense of myogenesis. DESIGN/METHODS: After Cytofluorimetric analysis of
cell surface markers, distinct MMP populations were sorted using specific
magnetic microbead associated antibodies and their ability to differentiate in
vitro in adypocytes and myotubes was assessed. Populations of interest underwent
in vivo studies conducted in immunodeficient Rag2-/- gc-/-
mice. RESULTS: The stem cell marker CD34 allowed us to sort two distinct
populations from human paediatric and adult muscle biopsies: CD34 positive (CD34+)
and CD34 negative cells (CD34-). In vitro, the CD34+ cells were myogenic and
adipogenic whereas the CD34- cells were only myogenic. Muscle regeneration
potential after transplantation in cryo-injuried muscle of immunodeficient Rag2-/-
gc-/- mice was the same for CD34+ and CD34- cells. To our
knowledge, there is no mouse model mimicking fibro-adipose degeneration typical
of human muscular dystrophies. Using clodronate-containing liposomes we could
obtain adipose degeneration in cryo-injured tibialis anterior muscle of Rag2/gc-/-
mice. After transplantation in these mice model CD34-cells were shown to
participate solely to muscle regeneration while CD34+ cells were driven
partially to differentiate into adipocytes. CONCLUSIONS/RELEVANCE: In
conclusion, muscle derived CD34 negative population might represent a new tool
for cell therapy of muscular dystrophy because it is committed toward myogenic
differentiation even in micro- environment permissive for fat development
[P06.099] Volumeteric Radial and Tibial Bone
Mineral Density in Boys with Duchenne Muscular Dystrophy
Wendy King, John Landoll, Velimir Matkovic, John T. Kissel, Columbus,
OH
OBJECTIVE: To examine volumetric bone mineral density (BoMD) in boys with
Duchenne muscular dystrophy (DMD). BACKGROUND: Whether patients with DMD
require bone modifying medications (e.g., bisphosphonates) to mitigate an
apparent decreased BoMD is uncertain. Patients with DMD do have lower areal BoMD
by DEXA compared to controls, but whether this reflects a true deficit in BoMD,
or rather is due to geometric parameters inherent in areal bone density analyses
is unclear. DESIGN/METHODS: We examined volumetric BoMD of the
non-dominant radius and tibia at distal (4%) and more proximal (33%) sites in
DMD boys and age-matched healthy male controls (n=8 pairs, age 10-20 yr, average
15.2) using a Stratec XCT-2000 peripheral quantitiative CT (pQCT) densitometer.
Results from DMD boys were compared to controls. RESULTS: Average total
cross-sectional area of the radius and tibia was significantly lower in DMD boys
compared to controls at both the distal (p=0.03) and proximal (p<0.001) sites.
The average total BoMD at the distal radius (p=0.004) and tibia (p<0.001) as
well as trabecular BoMD at the distal radius and tibia in DMD was also
significantly lower (p<0.001) than in controls. In both radius and tibia,
average cortical density at the proximal site was higher than that for controls
and was significant for the tibia (p<0.001). The relationships in volumetric
density between DMD and controls were consistent for both the radius and the
tibia. CONCLUSIONS/RELEVANCE: Boys with DMD have smaller bones than
age-matched controls. There is a real deficit in volumetric BoMD of the distal
forearm and tibia (trabecular bone) in boys with DMD. However, at more proximal
sites (representing cortical bone) BoMD is actually normal. These results
suggest that the relationship between BoMD, bone fragility and fracture risk in
boys with DMD may be more complex than previously appreciated, and that fracture
preventive measures may require more than simple bisphosphonate therapy.
[P06.098] A Comparison of the Six-Minute Walk Test
to Commonly Used Outcome Measures in Duchenne Muscular Dystrophy
Cassie J. Spalding, Erik K. Henricson, Craig M. McDonald, Richard T.
Abresch, Jay Han, Alina Nicorici, Leone Atkinson, Allen Reha, Gary Elfring,
Langdon Miller, Sacramento, CA, South Plainfield, NJ
OBJECTIVE: To evaluate the correlations between the 6-minute walk
distance (6MWD) and other commonly used measures of disease severity in Duchenne
Muscular Dystrophy (DMD). BACKGROUND: The lack of functional outcome
measures is an obstacle to the clinical development of new treatments for DMD.
The 6MWT has been used extensively in other disease states to measure global
physical functioning and is being used in DMD clinical trials. DESIGN/METHODS:
We enrolled ambulatory boys with DMD (n= 17) and able bodied control boys (CTRL)
(n=34) ages 4-12 years old. Outcome measures included a modified 6MWT, timed
motor function tests, Brooke & Vignos motor function scales, and isometric
quantitative strength testing. RESULTS: There was a significant
difference in outcome measure values between DMD and CTRL groups (p<0.001). In
DMD, 6MWD had a strong negative correlation with time to climb 4 stairs, time to
run/walk 10 meters, time to run/walk 25 meters (r=-0.7 to -0.9), and was
strongly correlated with Vignos lower extremity functional scale (r= -0.72).
6MWD had a moderate negative correlation (r=-0.45-0.69) with time to stand from
supine position, and a moderate correlation with the Brooke upper extremity
functional scale, and quantitative knee extensor strength. The 6MWD did not
correlate with quantitative knee flexor strength. Correlations between 6MWD and
other measures were not as strong in the CTRL group. CONCLUSIONS/RELEVANCE:
6MWD shows moderate to strong correlation with timed measures of physical
function in DMD. 6MWD shows a strong correlation with the Vignos lower extremity
motor function scale and a moderate correlation with the Brooke upper extremity
motor function scale. The 6MWD correlates moderately with isometric knee
extensor strength but not with knee flexor strength. The 6MWD measures global
physical function and endurance in DMD and provides a quantitative outcome
measure that is related to but different from commonly used measures of strength
and functional ability.
[P06.093] Oral Protein Supplementation Improves
Protein Metabolism after Acute Exercise in Patients with Fascio-Scapulo-Humeral
Muscular Dystrophy
Mette Cathrine Orngreen, Nicolai Preisler, Grete Andersen, Tina D.
Jeppesen, Gerrit Van Hall, John Vissing, Copenhagen, Denmark
OBJECTIVE: Investigating the effect of an acute bout of exercise on
skeletal muscle protein metabolism with and without protein supplementation in
patients with Fascioscapulohumeral muscular dystrophy (FSHD). BACKGROUND:
It has been shown that patients with FSHD can benefit from a 12-week,
low-intensity aerobic exercise program. However, it is unknown how skeletal
muscle protein metabolism of FSHD patients responds to an acute exercise bout,
and whether the FSHD patients can benefit from oral protein supplementation
immediately after exercise, by causing a net muscle protein gain as shown in
healthy controls. DESIGN/METHODS: Protein metabolism at rest, during 40
min of submaximal exercise, and three hours after exercise was studied in 3
patients with FSHD and 3 healthy subjects, using arterio-venous differences
across the leg, and L-[ring-2H5]phenylalanine dilution methodology.
All subjects received a protein drink right after the exercise bout in the
second trial. RESULTS: Protein supplementation induced a net protein
uptake (synthesis) in the resting period after exercise in FSHD and healthy
subjects, as compared to a net protein release (breakdown) without protein
supplementation (FSHD: -10437
vs.6717
and CTR: -12728
vs.281180
mmol/min). In accordance with this, arterial phenylalanine concentrations were
higher at rest after protein supplementation in both groups. CONCLUSIONS/RELEVANCE:
This study shows that net protein balance after an acute exercise bout is
comparable to that in healthy subjects, and shows that protein supplementation
boosts protein build-up after exercise. This indicates that the known beneficial
effect of aerobic training in FSHD, can potentially be enhanced by protein
supplementation after exercise training. Future controlled trials are warranted
to demonstrate this.
[S25.002] Pulmonary Function Characteristics of
255 Boys with Becker and Duchenne Muscular Dystrophy by Age Groups, Ambulatory
Status and Steroid Use: Baseline Data from the CINRG Longitudinal Study Project
Richard Abresch, Craig M. McDonald, Erik K. Henricson, Jay Han,
Robert Leshner, Diana Escolar, Eric Hoffman, Avital Cnaan, Adrienne Arrieta,
Fengming Hu, Angela Zimmerman, Tina Duong, Jill Mayhew, Julaine Florence, Davis,
CA, Sacramento, CA, Mc Lean, VA, Washington, DC, Saint Louis, MO
OBJECTIVE: To describe the pulmonary function characteristics of
ambulatory and non-ambulatory subjects with Becker and Duchenne Muscular
Dystrophy (B/DMD) and to determine whether steroid therapy is associated with
improvements in respiratory function. BACKGROUND: Several recent
conferences have reported that the lack of adequate natural history data and
well-characterized outcome measures limits the ability to conduct therapeutic
clinical trials in B/DMD. DESIGN/METHODS: Baseline pulmonary function
tests (PFTs) were performed in 255 subjects with confirmed B/DMD (aged 6 to 28
years) in 20 centers from 10 countries of the Cooperative International
Neuromuscular Research Group (CINRG). Pulmonary function measures included
forced vital capacity (FVC, %FVC), forced expiratory volume in 1 second (FEV1, %FEV1),
peak expiratory flow rate (PEFR, %PEFR), maximum inspiratory pressure (MIP),
maximum expiratory pressure (MEP), and peak cough flow (PCF). The PFT measures
were evaluated by current corticosteroid use, age groups (<10, 10-14, >15), and
ambulatory status (ambulatory, part-time wheelchair user, full-time wheelchair
user). RESULTS: All PFT measures except PCF were significantly different
in the 3 age groups (p<0.02). All PFT measures were significantly different when
compared to ambulatory status (p<0.01). All PFT measures except FVC, FEV1, and
PEFR were significantly higher in steroid users than non-users (p< 0.01).
Overall, all measures are most highly correlated in the oldest age group and
least correlated in the youngest group. FEV1 and FVC are strongly correlated
across age and ambulatory status (r = 0.9
0.99). PEFR is also fairly strongly correlated with FEV1 (r = 0.770.92).
PCEF is the least correlated with the other outcomes (r= 0.16- 0.71).
CONCLUSIONS/RELEVANCE: Pulmonary function measures clearly differentiate
between D/BMD patients of different ages and ambulatory abilities. Steroid use
significantly affects performance on pulmonary function measures suggesting that
PFTs are a suitable outcome measure for clinical trials and will help determine
the sample sizes needed.
[P07.098] Clinical Outcomes in Patients with
Duchenne Muscular Dystrophy and Role of Multidisciplinary Care
Muddasir Qureshi, Laurie Bliss, Brian Tseng, Charlestown, MA
OBJECTIVE: We sought to review clinical data in patients with duchenne
muscular dytrophy (DMD) seen at the Massachusetts General Hospital (MGH) and
assess role of multi-disciplinary care. Multidisciplinary care has been
initiated at the Pediatric Neuromuscular Unit at the MGH in June 2007.
BACKGROUND: Duchenne muscular dystrophy is a rare, progressive neuromuscular
disorder in boys leading to severe disability and death in adulthood. DESIGN/METHODS:
A retrospective study of people with DMD seen at the MGH was performed. All
participants were identified randomly using the MGH Research Patient Data
Registry (RPDR). Participants were selected if they had a confirmed diagnosis of
DMD. The data obtained included factors that may affect outcomes and clinical
care in patients with DMD including outpatient and emergency consult services,
hospitalizations, medication usage, pertinent laboratory values and incidence of
important co-morbidities. A univariate analysis was used to analyze the data (Fisher's
Exact Test, two-tailed, significance of p<0.05). RESULTS: A total of 43
patients with DMD were identified; 23 patients prior to and 20 patients after
the inauguration of multidisciplinary clinic at MGH in June 2007. Patients who
were seen after the start of multidisciplinary Pediatric Neuromuscular Clinic at
the MGH were more likely to attend an outpatient consult for cardiology
(p=0.0002), pulmonology (p=0.0001), endocrinology (p=0.0001), nutrition
(p=0.0001), physical therapy (p=0.03) and social work (p=0.0001). Prior to the
start of multidisciplinary care, approximately 25% of patients with DMD
presented to the emergency room with respiratory complaints without a previous
outpatient pulmonology consult. The incidence of cardiomyopathy (p=0.33) was
same in both groups however patients seen prior to June 2007 were more likely to
have scoliosis (p=0.01). Patients treated at the multidisciplinary clinic were
more likely to receive steroid therapy (p=0.03). CONCLUSIONS/RELEVANCE:
Multidisciplinary care in boys with DMD is more likely to prevent the various
complications that may arise in this patient population.
P06.104] An Immortalized Fibroblast Model for
Exon-Skipping Studies in Duchenne Muscular Dystrophy (DMD)
Ling Zhao, Gillian Butler-Browne, Vincent Mouly, Kamel Mamchaoui,
Steve Wilton, Kevin M. Flanigan, Paris, France, Nedlands, Western Australia,
Australia, Salt Lake City, UT
OBJECTIVE: To develop a novel immortalized cell line derived from a
patient with a duplication of exons 8 and 9 in the DMD gene as a tool for
exon skipping studies. BACKGROUND: AON-mediated exon skipping
demonstrates significant potential as a therapy for DMD. Fibroblasts require
MyoD induced transdifferentiation in order to express significant amounts of
DMD mRNA. DESIGN/METHODS: A primary fibroblast cell line was
immortalized via lentiviral transfection with the human telomerase gene, and
subsequently transfected with a lentivirus carrying the MyoD gene under the
control of a tetracycline-inducible promoter. After growth to near confluence,
cells were switched from growth to differentiation medium containing doxycyline
(2 ug/ml), and treated with AONs directed toward exon 8, exon 9, or both exons 8
& 9 in both time course and dose escalation experiments. Dystrophin expression
was analyzed by immunofluorescence (days 1, 3, 5, and 7) and immunoblot (days 5
and 7). RESULTS: tet-MyoD fibroblast line successfully
transdifferentiates to a myoblast lineage as judged by expression of desmin and
alpha-actinin. RT-PCR studies from untreated cells reveal some baseline exon
skipping, resulting in both wild-type and duplication-containing mRNA. AONs
directed toward exons 8 and 9, or exon 9 alone, significantly increase the
degree of skipping, whereas treatment with the AON directed toward exon 8 alone
induces no significant skipping. Treatment at 100mM results in an increased
dystrophin expression, and immunoblot reveals expression of dystrophin protein
of apparently normal size. CONCLUSIONS/RELEVANCE: A tet-MyoD fibroblast
line is a valid model for AON-mediated exon skipping studies. Duplication of
exons 8 and 9 may be amenable to therapy with AONs, resulting in wild type
dystrophin protein expression.
P06.090] Cystatin C Permits Assessment of Renal
Glomerular Filtration Independant of Muscle Mass in Duchenne Muscular Dystrophy
(DMD)
Susan Gailey, Cheryl Wall, Laurence Viollet, David Thornton, Jerry
R. Mendell, Columbus, OH
OBJECTIVE: Establish a laboratory test for assessment of renal function
in DMD independent of muscle mass BACKGROUND: Assessing renal function
using standard laboratory tests represents a major challenge in DMD and other
dystrophies where muscle mass is severely reduced. Serum creatinine, a
derivative of skeletal muscle creatine, is inadequate for monitoring renal
function. Creatinine clearance (CrCL), often considered the gold standard for
renal glomerular filtration suffers the same limitations being similarly
dependent on muscle derived creatine. Iohexol infusion has been suggested as an
alternative to CrCL, but measurement requires prolonged infusion with multiple
blood draws, and results are not available for 4-6 weeks. Cystatin C is a
nonglycosylated protein that is unrelated to muscle mass making it potentially
an ideal gauge of renal function for DMD patients. Testing new therapeutic
agents mandates validating an alternative practical approach for measuring renal
function. DESIGN/METHODS: Ninety subjects were recruited for this study
including three cohorts of 30 subjects each, 5-20 years of age. Thirty subjects
had mutation proven DMD, 30 subjects were healthy controls, and 30 subjects had
known renal disease. The DMD population was balanced for corticosteroid use and
ambulation. RESULTS: Cystatin C levels in the DMD population were within
established normal reference range and varied from 0.5-0.9 mg/L. The mean (+
SD) cystatin C for 30 DMD patients was 0.693 + 0.122 compared to normal
controls 0.693 + 0.083 and subjects affected with renal disease 3.517
+ 2.142. There was no difference between DMD and normals, while the renal
disease patients differed from both of the others (p<0.001). Neither
corticosteroid use, ambulation or age affected Cystatin C in the DMD population.
CONCLUSIONS/RELEVANCE: This is the first study validating cystatin C as
an alternative and important means of monitoring renal function in DMD. Cystatin
C has particular applicability for testing novel agents with potential renal
toxicity.
[P06.100] Newborn Screening (NBS) in Duchenne
Muscular Dystrophy: A Practical, Comprehensive and Affordable Approach
Roula Al-Dahhak, Susan Gailey, Kandice Roush, Lauren Schueller, Joni
Beauman, Heather Lilly, Sharon Linard, Ram Chandrasekar, Diane Dunn, Deborah
Cragun, Christopher Shilling, Patrick Wall, Nancy Leslie, Natalie Street, Robert
Weiss, Jerry Mendell, Reynoldsburg, OH, Cincinnati, OH, Atlanta, GA, Salt Lake
City, UT, Columbus, OH
OBJECTIVE: To establish a comprehensive, practical, affordable approach
to NBS for DMD BACKGROUND: DMD, caused by mutations of the dystrophin (DYS)
gene, is the most common severe form of childhood MD. DYS, the largest gene
identified to date, exhibits a very high mutation rate, estimated to occur in
1/10,000 germ cells. This means that the disease can never be eliminated,
emphasizing two important issues related to NBS. First, considering that most
DMD cases go undiagnosed until age 3 or later, the possibility of additional
affected offspring is very high unless there is detection of the first born
male. Secondly, translational research efforts emphasize the value of early
intervention, that is best ensured by early detection. In this study, we
developed an approach to NBS not previously used, permitting creatine kinase (CK)
screening and DNA analysis on the same dried blood spot (DBS) set taken at birth.
DESIGN/METHODS: CKs were measured on one of the DBS obtained at birth.
Based on sampling of 25,000 anonymous newborn males, CKs >600U/L prompted DNA
testing on a second DBS using SCAIP, single condition amplification/internal
primer sequencing. RESULTS: To date 7000 newborns have had CK screening
(95 fell between 600-999 U/L, 7 between 1000-1499 U/L, 1 between 1500-1999 U/L).
Two were >2000 U/L (2461 and 2675 U/L) and DYS mutations were found in both
cases. The project is ongoing throughout the State of Ohio and will provide
additional results at the time of presentation. CONCLUSIONS/RELEVANCE:
This study demonstrates a practical, affordable, and comprehensive approach to
conducting NBS for DMD. DNA testing can be limited to DBS exhibiting the highest
level CKs, at a cost of
$1.00/sample.
Of interest, our findings in this outbred population continue to support an
incidence of DMD in
1:3500
newborns. Further data obtained through expansion to the statewide program will
add to the accuracy of these estimates.
[P06.103] Abundance of Circulating Progenitors
with Myo-Endothelial Potential Correlates with a Mild Phenotype in Patients
Affected by Duchenne Muscular Dystrophy
Marzia Belicchi, Andrea Farini, Mirella Meregalli, Daniele Parolini,
Paola Razini, Raffaella Lopa, Laura Porretti, Simona Maciotta, Claire Navarro,
Giulio Cossu, Nereo Bresolin, Yvan Torrente, Milan, Italy
OBJECTIVE: We hypothesized that the levels of circulating stem cells
expressing the CD133 antigen which possess myo/endothelial potential would
predict the progression of DMD. We tried to find a subpopulation of CD133+ stem
cells significantly higher in DMD patients compared with healthy controls and
positively correlated with the clinical score. BACKGROUND: We
hypothesized that the levels of circulating stem cells expressing the CD133
antigen which possess myo/endothelial potential would predict the progression of
DMD. We tried to find a subpopulation of CD133+ stem cells significantly higher
in DMD patients compared with healthy controls and positively correlated with
the clinical score. DESIGN/METHODS: The levels of several subpopulations
of circulating stem cells expressing the CD133 antigen were determined by flow
cytometry in 70 DMD patients. The median (6SD) age of the population was
10.6663.81 (range 3 to 20 years). The levels of the subpopulation of the CD133+
stem cells were correlated with the clinical condition. Samples were obtained
from routine blood tests performed in healthy and DMD subjects. RESULTS:
The levels of CD133+CXCR4+CD34- stem cells were significantly higher in DMD
patients compared to healthy controls with a tendency towards decreased levels
in older patients. In a subgroup of 19 DMD patients after 24 months of
follow-up, increased levels of CD133+CXCR4+CD34- cells was shown to be
associated with a phenotype characterised by slower disease progression.
CONCLUSIONS/RELEVANCE: Linear regression analysis showed a direct
correlation between the levels of circulating CD133+CXCR4+CD34- and the clinical
condition of the DMD patients. Based on these data we believe that the levels of
a subpopulation of circulating CD133+ stem cells in DMD patients may be a
promising new prognostic clinical marker of the progression of the disease with
practical significance to allow any beneficial effect in future clinical trials.
[P06.102] Characterization of DMD Mutations
in Novel Dog Models of Dystrophinopathy
Kevin M. Flanigan, Aaron Larsen, Laura Taylor, Janet Bogan, Daniel
Bogan, Joe Kornegay, Salt Lake City, UT, Chapel Hill, NC
OBJECTIVE: To characterize DMD mutations in novel dog models of
Duchenne muscular dystrophy. BACKGROUND: Duchenne muscular dystrophy
(DMD) and Becker muscular dystrophy (BMD) are X-linked disorders caused by
mutations in the DMD gene. Several dystrophic dogs have been identified
in clinical veterinary practice by the presence of variable weakness and altered
or absent dystrophin expression. Genomic studies to define causative mutations
have not been done in most of these dogs. Here, we describe mutations in a
Labrador retriever with severe weakness and a cocker spaniel with milder
weakness. Carrier status was also documented in a Yorkshire terrier related to a
dog known to have a deletion of exons 3-7. DESIGN/METHODS:
Immunofluorescent and immunoblot analysis of skeletal muscle was performed using
a panel of anti-dystrophin antibodies. For novel mutations, sequencing of the
complete DMD cDNA (generated via reverse transcriptase-PCR from
muscle-derived mRNA), and mutations confirmed by sequence analysis from genomic
DNA. For detection of deletion 3-7 carrier status, quantitative real-time PCR (qRT-PCR)
was performed on an ABI 7500 HT, comparing the amplification curve for exon 3 to
that of exon 49 via standard methods. RESULTS: Two novel mutations were
found. In the Labrador retriever, we identified a LINE element insertion,
resulting in the inclusion of a pseudoexon within the mRNA. In the cocker
spaniel, we identified a 4 nucleotide frameshifting deletion in exon 65,
resulting in a premature stop codon at the site of the frameshift. In the
Yorkshire terrier, qRT-PCR robustly identified the carrier status.
CONCLUSIONS/RELEVANCE: Dystrophic dogs are important large animal models of
human DMD that can be used to study research questions not addressable in the
standard mdx mouse model. Identification of these novel mutations could
facilitate preclinical studies directed toward these specific mutation classes.
[P06.087] Reduced Necrosis and Inflammatory Change
in Muscle of SJL/J Mice by Blocking TNFa Function with Etanercept
Hiroshi Nemoto, Shingo Konno, Hideki Sugimoto, Nobuatsu Nomoto,
Hiroshi Nakazora, Mayumi Murata, Toshiki Fujioka, Tokyo, Japan
OBJECTIVE: In SJL/J mice the damage of sarcolemma may relate the
inflammatory change, especially cytokines. We test the hypothesis that TNF alpha
contribute the myofibril necrosis. In this study, we examine the histological
and immunohistological changes of the muscle of SJL/J mice by the treatment of
etanercept. BACKGROUND: Dysferlin has the function to repair the muscle
membrane from mechanical stress. SJL/J mice show the defect of dysferlin, it
believes that SJL mice are an animal model of Limb-girdle type muscular
dystrophy 2B and Miyoshi myopathy. Also older SJL/J mice show the inflammatory
changes in skeletal muscle. The mechanism of inflammation in muscle may be
related immunological abnormality. Anti TNF alpha therapy for mdx mice that are
the model mice of Duchenne type muscular dystrophy is effective. DESIGN/METHODS:
Sixty-day old mice are injected 30ug/kg of etanercept subcutaneously twice per
week until sacrificed. Control mice are injected 0.1% of manitol solution
subcutaneously. Mice are sacrificed at day 120, 240, 360(n=5) and 420 (n=3) in
each group. Muscle samples were collected from biceps and quadriceps femoris.
Histlogical and immunohistlogical study (CD11b, CD4, CD8) are performed
RESULTS: Sixty-day old mice are injected 30ug/kg of etanercept
subcutaneously twice per week until sacrificed. Control mice are injected 0.1%
of manitol solution subcutaneously. Mice are sacrificed at day 120, 240,
360(n=5) and 420 (n=3) in each group. Muscle samples were collected from biceps
and quadriceps femoris. Histlogical and immunohistlogical study (CD11b, CD4,
CD8) are performed. CONCLUSIONS/RELEVANCE: As a result of blocking TNF a
by entercept, it makes reduction of the activity of macrophage. It makes the
suppression of the activity of inflammatory changes and also the damage of
muscle. In this study we present the suppression of degenerative and
inflammatory changes of older SJL/J mice treated by etanercept. TNF a may
contribute to the breakdown of dysferinopathy muscle.
[P06.094] Partial Response of Dysferlinopathy
Patients to Low Dose Dantrolene Sodium Treatment
Itzik Wirguin, Beer-Sheva, Israel
OBJECTIVE: To describe a modest ameliorating effect of Dantrolene Sodium
(DS) on the progression of Limb Girdle Muscular Dystrophy 2B (LGMD2b) in 2
siblings. BACKGROUND: LGMD2b, Miyoshy myopathy (MM) and distal anterior
compartment myopathy are autosomal recessive progressive muscular disorders
caused by mutations in the Dysferlin gene. Dysferlin protein is involved in the
calcium dependent membrane fusion step of the muscle fiber repair process. Two
clusters of dysferlinopathy were described in Israel, of which one is caused by
a frameshift mutation of G deletion at codon 927 (2779delG, Neuromuscular
Disorders 2007:17:950-954). DS is an agent used in the treatment of malignant
hyperthermia and to relieve spasticity, probably through its ability to block
calcium release from the sarcoplasmic reticulum. A recent report suggested its
effect in a compound heterozygous variant of MM in Japan. DESIGN/METHODS:
Two siblings, homozygous for the 2779delG mutation presented with progressive
weakness (MM phenotype) around the age of 15. CK levels were around 14000 in the
older male and around 7000 in the younger female. Both patients were treated
with DS, 25 mg once daily and monitored for systemic toxicity, clinical status
and muscle power. RESULTS: Both patients reported significant improvement
in exercise tolerance, ability to work and self perceived life quality, within 1
week of treatment initiation. CK levels declined by 20-25% and compound muscle
chart scores rose by about 3 points. Blood tests showed no change in liver
enzyme levels. At the time of this report, treatment has been given for 6 months
during which no clinical deterioration has occurred. A short term attempt to
raise the dose to 25 mg BID brought no additional benefit. CONCLUSIONS/RELEVANCE:
DS has a discernible positive effect in patients with MM type dysferlinopathy.
These results warrant a randomized clinical trial of DS in a wider range of
patients with Dysferlin mutations.
[SC02.003] Uptake and Outcome of Genetic Testing
among Participants of the DuchenneConnect Registry
Vanessa Rangel Miller, Giovanna Spinella, Ken Loud, W. Andrew
Faucett, Madhuri Hegde, Pat Furlong, Falls Church, VA, Atlanta, GA, Middletown,
OH
OBJECTIVE: DuchenneConnect is a self-report patient registry developed to
connect patients, providers, and research communities. The program promotes
access to genetic testing, genetic counseling, and resources for clinical trials,
research studies, clinical care, and management. This report describes the
frequency of genetic testing, informative test results, and further needs among
participants. BACKGROUND: In Duchenne/Becker muscular dystrophy (DBMD),
well-characterized mutations are necessary to evaluate eligibility for
therapeutic trials using molecular genetic corrective approaches. Despite
advances in genetic testing, many patients do not have results that provide
well-characterized mutation information. DESIGN/METHODS: In November
2007, we developed a web-based registry to capture patients' clinical
presentation and DMD mutation. Design of the survey and review of the
data follows guidelines employed by other North American DBMD registries and the
TREAT-NMD Neuromuscular Network. A printable report tool enables participants to
have their medical care provider review and verify the registry information.
RESULTS: As of October 2008, 918 of 1,234 (74%) registrants completed a
survey: 501(55%) had genetic testing, 267(29%) had a muscle biopsy plus genetic
testing, 65(7%) had a muscle biopsy alone, 53(5.5%) did not have testing, and
16(1.7%) responded 'I don't know'. From review of 146 of 293 genetic test
results: 66(45%) were informative,
31(21%)
warranted additional testing,
31(21%)
used methods not recommended by the international registry, and 17(12%) were
research results needing clinical laboratory confirmation. 812 requests were
made to speak with a genetic counselor, including: 291(36%) to discuss genetic
testing and/or results, 145(21%) to discuss the registration process, and
77(9.5%) to arrange genetic testing. CONCLUSIONS/RELEVANCE: A substantial
number of participants require further genetic testing due to insufficient
mutation characterization. Full mutation characterization requires
identification of the boundaries of a deletion/duplication or a deleterious
point mutation. Comprehensive dystrophin testing provides information essential
for participation in clinical trials, therapeutic decision-making, accurate risk
counseling, and testing for family members.
[P08.117] Phase I Gene Transfer Clinical Trial in
Alpha-Sarcoglycan Deficiency (LGMD2D)
Jerry R. Mendell, Louise R. Rodino-Klapac, Xiomara Rosales-Quintero,
Brian D. Coley, Zarife Sahenk, Vinod Malik, Jo Craenen, Sarah E. Lewis, Brent
Yetter, Katie J. Campbell, William G. Bremer, Barry J. Byrne, Christopher
Walker, K. Reed Clark, Gainesville, FL, Columbus, OH
OBJECTIVE: Alpha-SG gene (SCGA) replacement is a potential treatment
strategy for limb girdle muscular dystrophy (LGMD)2D. BACKGROUND: There
is no treatment for LGMD2D caused by SCGA deficiency. Adeno-associated virus (AAV)
mediated gene replacement appears to be a viable option based on pre-clinical
studies in knock out mice. AAV1, using a muscle specific promoter to avoid off
target effects, mediates persistent gene expression with a favorable safety
profile. DESIGN/METHODS: Under conscious sedation, LGMD2D patients (ages
12 and 10) underwent gene transfer using a double-blind protocol with injections
bilaterally to the extensor digitorum brevis (EDB) muscles. One side received
only saline while the other received AAV1.tMCK.SGCA, 3.25 X1011 vg in
1.5 ml. Intramuscular injections were guided by ultrasound and EMG to ensure
proper needle placement for gene transfer. RESULTS: Thus far, two LGMD2D
subjects have received gene transfer without adverse effects. There was no
swelling or erythema at the site of injection and no systemic reaction following
gene transfer. In one subject the EDB muscles on both sides were removed at day
43 post gene transfer and analyzed without breaking the code. Muscle from left
and right EDBs were compared for gene expression using western blots and direct
immunofluorescence (IF). A three fold increase in gene expression was seen on
the left side. A second case underwent a similar procedure and reached higher
levels of gene expression on the right side at three months post gene transfer.
Presence of transgene DNA was detected by PCR on the side corresponding to gene
expression in both patients. There were no clearly defined immune responses to
AAV1 or transgene product. CONCLUSIONS/RELEVANCE: Gene transfer is
possible in the injected muscle of LGMD2D patients but the clinical significance
and long-term safety require further study. Additional patients will be enrolled
(goal= 6 subjects) and will include careful scrutiny of mononuclear cells in
muscle.
[P06.101] Sonoporation of Human Biceps Muscle as
Preparation for Plasmid-Based Dystrophin Gene Transfer
Maria J. Molnar, Renald Gilbert, Aniko Gal, George Karpati, Budapest,
Hungary, Montreal, QC, Canada
OBJECTIVE: To determine the feasibility and safety of sonoporation (SP)
in biceps muscles of 5 healthy volunteers in Phase I/A clinical trial. in
preparation for plasmid-mediated dystrophin gene transfer in Duchenne muscular
dystrophy (DMD) BACKGROUND: Gene transfer is a proven therapeutic
modality in genetic diseases such as DMD. Gene transfer using plasmids as
vectors is much less problematic than viral vectors. In mdx mice we have
demonstrated that plasmid containing a full length dystrophin cDNA was efficient
as long as it was coupled with SP (Mol Ther 6:687-693, 2002). SP consists of
injection into muscle a suspension of microspheres filled with an inert gas
which will burst from the heat generated by SP. This, in turn, creates
reversible microgaps at the surface membrane of the muscle fibers through which
the plasmid particles can stream into the muscle fibers. DESIGN/METHODS:
After signing informed consents, five healthy volunteers (4 males/1 female. aged
25-35 years) had their left biceps muscles injected with 1.5 ml of microsphere
solution (DEFINITYR). The right biceps was injected with saline.
Immediately after, ultrasound was delivered percutaneously over a 2 minute
period to both muscles from a custom-designed sonoporator (1 MHz, 2-3 w/cm/2,
30% duty cycle).Thirty six hours later a muscle biopsy was studied with
histochemistry, confocal and electron microscopy. RESULTS: In 2 subjects,
transient reddening of the skin at the sonoporation site was present. There were
a few scattered endomysial mononuclear inflammatory cell infiltrates, but no
partial invasion or necrosis of muscle fibers or apoptotic myonuclei were
observed. Subsarcolemmal vesicular profiles were conspicuous. A few short
sarclolemmal gaps were still present. CONCLUSIONS/RELEVANCE: Sonoporation
of human muscles is feasible and safe as it produced no significant muscle
damage. It is expected to highly facilitate the efficiency of plasmid-mediated
therapeutic gene transfer in DMD.
[P06.092] LGMD 2I in Twin Brothers: Response to
Prednisone
Peter I. Karachunski, Marcia Margolis, Joline Dalton, John W. Day,
Minneapolis, MN
OBJECTIVE: We studied monozygotic twins with limb-girdle muscular
dystrophy type 2I (LGMD-2I) who were haploinsufficient for fukitin-related
protein (FKRP) containing the L276I mutation. They had episodic weakness that
became progressive and unremitting. We used quantitative motor function to
determine the effects of steroids. BACKGROUND: LGMD-2I is caused by
mutations in FKRP, a putative glycosyltransferase that is involved in
a-dystroglycan glycosylation. LGMD-2I can present with various phenotypes based
on the age of onset. A form of LGMD-2I has been described in children developing
weakness subacutely, in a time frame comparable to viral myositis. Anecdotal
reports have suggested LGMD-2I responds to steroids. DESIGN/METHODS: We
assessed 9 year old twin brothers every 4 to 8 weeks over 18 months in a
cross-over study. We measured motor function using handheld dynamometry, manual
muscle testing, a Step Watch Activity Monitor, timed function tests (walking 10
m, climbing stairs, and standing from supine) and pulmonary function testing.
Twins were assessed prior to treating first one, then the other, with prednisone
(0.75mg/kg daily), for 6 months. RESULTS: Episodes of severe weakness did
not occur during period of observation in either subject. Muscle strength and
function improved while each boy was on prednisone, and declined when prednisone
was discontinued in twin A. Forced vital capacity (FVC) improved from 80% to
100% of predicted in twin A but returned to baseline when prednisone was
discontinued. FVC remained stable in twin B on steroids. Manual motor testing
and handheld dynamometry showed improvement on prednisone but with greater
inter-test variability. Weight gain and cushingoid appearance developed on
steroids, without correlation with decline in performance. CONCLUSIONS/RELEVANCE:
This study expanded description the recognized phenotype of LGMD-2I including
infantile onset of episodic weakness, and demonstrated a response to short term
prednisone. A larger study will be required to determine whether this treatment
ameliorates weakness in all LGMD-2I patients.
[P06.106] High-Resolution, Three-Dimensional,
In Vivo Bioimaging of Perivascular Stem Cell Homing
Chad D. Markert, Jennifer K. Cann, Martin K. Childers, Winston Salem,
NC
OBJECTIVE: To track labelled perivascular progenitor cells following
intramuscular or intra-arterial injection. BACKGROUND: Duchenne muscular
dystrophy (DMD) is a progressive, lethal, X-linked disease of skeletal and
cardiac muscle affecting nearly 1 in 3500 newborn males annually. DMD is caused
by mutation of the dystrophin gene, a vulnerable target for new mutations. The
cardiac and skeletal muscles of DMD patients are devoid of the dystrophin gene
product, a 427-kD protein called dystrophin. To address the progressive and
ultimately fatal degeneration of DMD muscles, intense research efforts are aimed
at tilting the balance in favor of regeneration. Stem cell transplantation is
one therapeutic approach. DESIGN/METHODS: Here we provide data using
several tracking methods (intravital microscopy, MRI, in vivo fluorescent
imaging, and immunohistochemistry) which allow us to follow the migration of
intra-arterially injected perivascular progenitor cells (pericytes) from the
point of their systemic injection to the point of their engraftment at a site of
skeletal muscle injury, where they are needed for muscle repair. RESULTS:
MRI scans of cardiotoxin-injured mice infused (femoral artery) with labeled
pericytes show 1) pericyte localization at areas of skeletal muscle injury and
2) accumulation of injected pericytes inside long bones of the hindlimb,
indicating that exogenous pericytes
home
to the bone marrow, a canonical mesenchymal stem cell niche. CONCLUSIONS/RELEVANCE:
Isolation of perivascular stem cells allows us to provocatively speculate that
these cells embody regenerative promise for the following reasons: first, they
are readily available in virtually every vascularized tissue, including term
placenta, which is a high-throughput, easily accessible source; second, they
have myogenic potential; third, they are conducive to systemic delivery; fourth,
they home to sites of injury efficiently, and fifth, experiments are underway to
characterize them based on other key properties making them appropriate for
cellular therapy of degenerative muscle disease. Importantly, unlike embryonic
stem cells, they do not engender ethical debate.
[P08.118] Restoration of Normal Function by
Combinational Gene Replacement Using Follistatin and Micro-Dystrophin in Mdx
Mice
Louise R. Rodino-Klapac, Paul M. L. Janssen, Kim M. Shontz, Amy
Eagle, Chrystal L. Montgomery, K. Reed Clark, Jerry Mendell, Brian Kaspar,
Columbus, OH
OBJECTIVE: To normalize force generation and protect against eccentric
contraction by combinational gene replacement with follistatin and dystrophin
mini-genes. BACKGROUND: Duchenne muscular dystrophy is a devastating
disease characterized by severe muscle weakness due to lack of dystrophin. Gene
replacement using AAV, with its small insert capacity, forces truncation of the
dystrophin gene (mini-gene), resulting in a product that incompletely restores
muscle function. AAV delivery of follistatin, a known myostatin inhibitor,
increases muscle size and strength in dystrophic mdx mice (Haidet et al. 2008)
but leaves the sarcolemma vulnerable. Follistatin combined with micro-DYS is
strategically positioned to achieve functional restoration, both protecting the
fragile dystrophin-deficient sarcolemma and at the same time, normalizing force
generation. DESIGN/METHODS: Six-month old mdx mice were treated with
combinatorial gene therapy using rAAV.micro-DYS and FS344 (an isoform sparing
activin binding to protect the pituitary gonadal axis) by direct muscle
injection into the tibialis anterior/extensor digitorum (EDL) complex. At 1 year
these mice were studied for force generation and protection against eccentric
contraction and correlative morphology. RESULTS: rAAV.micro-DYS
incompletely improves force generation and partially protects against eccentric
contractions compared to untreated mdx, but not to wild-type levels. In contrast,
simultaneous delivery of AAV1.FS344/micro-DYS reaches wild-type maximum force
generation and resistance to damage by repeated eccentric contractions. Of
interest, FS344 can accomplish this task with treatment 3 months before or after
micro-DYS delivery. These findings correlated with improved hindlimb grip
strength and reversal of dystrophic features by histopathology. Complete
necropsies of mice undergoing gene therapy demonstrated no evidence of off
target affects. CONCLUSIONS/RELEVANCE: Although AAV gene therapy
treatment for DMD is limited by the inability to deliver full-length dystrophin,
combinational therapy with follistatin augments function, compensating for the
limitations of small dystrophin gene replacement. This approach expands
translational options.
[P09.092] A Cooperative International
Neuromuscular Research Group (CINRG) Study of the Relationship between
Impairment, Activity Limitation, Participation and Quality of Life in Persons
with Confirmed Dystrophinopathies: Methods and Baseline Characteristics
Erik K. Henricson, Craig M. McDonald, Richard T. Abresch, Jay J. Han,
Robert Leshner, Eric P. Hoffman, Diana Escolar, Avital Cnaan, Fengming Hu,
Angela Zimmerman, Tina Duong, Jill Mayhew, Julaine Florence, Adrienne Arrieta,
Sacramento, CA, Mc Lean, VA, Saint Louis, MO, Washington, DC
OBJECTIVE: To conduct the first large-scale longitudinal investigation of
severe Becker and Duchenne muscular dystrophy (B/DMD) that uses the World Health
Organization's International Classification of Functioning, Health and
Disability. BACKGROUND: Investigators studying therapeutics in B/DMD face
increasing pressure from regulatory authorities to develop sensitive, reliable
and meaningful outcome measures of strength, function, and person-reported
health-related quality of life (HRQoL). DESIGN/METHODS: We enrolled males
with confirmed B/DMD from 20 participating centers from 10 countries of the
Cooperative International Neuromuscular Research Group (CINRG). Following
consent and review of diagnostic testing, participants undergo standardized
assessments at baseline, months 3, 6, 9, 12, 18, 24, 36, 48 and 60. DNA is
collected for banking. Study teams collect age-appropriate measures of clinical
history (medical events, medications, durable medical equipment, supportive
services), anthropometrics, goniometry (wrists, elbows, knees, ankles), Medical
Research Council manual muscle strength, quantitative knee and elbow
flexor/extensor and hand grip strength using the CINRG Quantitative Measurement
System, timed function (stand from supine, stair climb, 10M walk/run), Brooke
and Vignos functional scales, pulmonary function (FVC, FEV1, MIP,
MEP) and health-related Quality of Life (including PedsQL, SF-36, POSNA).
RESULTS: Two hundred ninety males with B/DMD aged 2 to 28 years of age were
enrolled at 20 CINRG centers worldwide between Fall 2005 and Fall 2008.
Participants were well-represented from the youngest ages to the mid-teens, with
fewer individuals in their late teens to early twenties. At enrollment, 65% were
current corticosteroid users, 63% were fully or partially ambulatory and 13%
required some ventilatory assistance. We will present baseline assessment data
relative to age, ambulatory status and corticosteroid use. CONCLUSIONS/RELEVANCE:
This study provides investigators with data on age-related patterns of clinical,
functional and person-reported outcomes in individuals with B/DMD. This will
contribute to optimal design of future clinical trials of emerging therapeutics.
[P06.096] Creatine Kinase Elevation: Evaluation of
Pediatric Population
Sebastien Perreault, Ala Birca, Dominique Piper, France Gauvin,
Michel Vanasse, Montreal, QC, Canada
OBJECTIVE: To determine the etiologies and the clinical evolution of
children with creatine kinase (CK) elevation. BACKGROUND: In children, CK
elevation is caused by multiple diseases. In some cases, early identification of
underlying etiology can improve management DESIGN/METHODS: Retrospective
study conducted in a tertiary care university-affiliated pediatric hospital. All
consecutive patients from 2001 to 2002 with CK levels
1000
IU/L were included. Using a standardized form, we collected data from each
patient's charts. RESULTS: 143 patients were included in the study (males
57%; mean age: 7,5 years
5,9). The median elevation of CK was 2337 (range:1003-811428 IU/L). The most
frequent diagnosis were (n;%) : viral myositis (29;20%), trauma (24;17%),
surgery (24;17%), anoxia (12;8%), drug reaction (6;4%) and metabolic myopathies
(2 ;1,4%). In 17 patients (12%) no formal diagnosis could be made with certainty.
Few patients developed complications secondary to rhabdomyolisis. Two patients
had acute renal failure due to rhabdomyolisis. For one of them, delay in
diagnosing rhabdomyolisis probably contributed to the severity of the acute
renal failure. Chronic CK elevation was present in 13 patients, of which 3 had
dermatomyositis and 8 dystrophynopathies.In 4 cases of dystrophynopathy, the CK
elevation was identified incidentally, prior to clinical suspicion of muscle
disorder. Those cases were initially investigated for mental retardation or
hepatic enzyme elevation. CONCLUSIONS/RELEVANCE: Etiologies of CK
elevation in children are various and differ from those reported in adults. In
most patient, CK elevation is benign and presents no recurrency. In some cases,
rhabdomyolisis is the initial sign of a metabolic myopathy. For others cases
like dystrophynopathy, CK elevation is present even when there is no obvious
clinical manifestations.
[S25.003] Combining Stem Cells and Exon Skipping
and Approach To Treat Muscular Dystrophies
Mirella Meregalli, Andrea Farini, Marzia Belicchi, Rachid Benchaouir,
Giuseppe D'Antona, Daniele Parolini, Simona Maciotta, Paola Razini, Roberto
Bottinelli, Luis Garcia, Nereo Bresolin, Yvan Torrente, Pavia, Italy, Paris,
France, Milan, Italy
OBJECTIVE: The main goal of this work would be to combine gene
modification strategies with cell-mediated therapies. This approach could permit
the autologous transplantation of cells, minimizing the risk of implant
rejection BACKGROUND: Mutations in the dystrophin gene cause hereditary
muscular diseases as BMD and DMD. The characterization of dystrophin gene and
the evidence that different types of adult stem cells are capable of muscle
regeneration has lead to the development of potential gene therapy and stem cell
treatments for DMD. In some cases, forced exclusion (skipping) of a single or
multiple exons can restore the reading frame, giving rise to a shorter, but
still functional protein DESIGN/METHODS: Blood and muscle CD133+ were
collected from peripheral blood of normal or DMD patients and from skeletal
muscle biopsies respectively. The cells were transduced with lentiviral vectors
constructed to convey specific antisense oligonucleotides able to induce an
efficient exon-skipping and to correct the initial frameshift caused by the DMD
deletion. The skipped stem cells were injected in scid/mdx mice to verify the
human dystrophin expression and the capacity of the skipped cells to fuse with
regenerating muscle fibers. RESULTS: The skipped blood and muscle-derived
stem cells were able to fuse in vivo with scid/mdx mice regenerative fibers and
to perform expression of a functional human dystrophin and restructure the
dystrophin-associated complex. Muscle function was evaluated in vitro by
measuring tetanic force of isolated injected TA and EDL muscles and in vivo
by using treadmill exhaustion tests. CONCLUSIONS/RELEVANCE: Autologous
engrafting of blood or muscle-derived CD133+ cells, preliminary genetically
modified to re-express a functional dystrophin, seems to represent a promising
approach for DMD. This approach should offer the chance to distribute the
autologous corrected stem cells to the whole body musculature with an
intra-arterial injection providing a clinical benefit for the dystrophic
patients.