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2017 NEWS

MARCH

12 - Abstracts thar will be presented in American Academy of Neurology Meeting, April 2017

a) Therapeutic Effect of KPT-350 in a Preclinical Model of Duchenne Muscular Dystrophy

Devin Gibbs1, Rylie Hightower2, Christopher Lee1, Janelle Spinazzola1, Lillian Mead1, Jeffrey Widrick1, Sharon
Tamir3, Shelton Cochran3, Yosef Landesman3, Louis Kunkel1, Matthew Alexander4
1Division of Genetics and Genomics, Boston Children's Hospital and Harvard Medical School, 2University of
Alabama at Birmingham Graduate School of Biomedical Sciences, 3Karyopharm Therapeutics, 4Department of
Pediatrics, Division of Neurology at Children's of Alabama and the University of Alabama at Birmingham

Objective:
This study evaluated the effectiveness of the Selective Inhibitor of Nuclear Export (SINE) compound KPT-350 in a zebrafish model of Duchenne muscular dystrophy (DMD).

Background:
DMD is an X-linked disorder that afflicts approximately 1:5000 live male births, making it the most common form of muscular dystrophy worldwide. The nuclear export protein XPO1/CRM1 is a promising target for the treatment of neurological disorders with inflammatory pathology such as DMD. IκBα, a protein cargo of XPO1, inhibits proinflammatory transcription factor NF-κB activity by preventing its transcription. KPT-350 is a potent, orally available, slowly reversible, small molecule inhibitor of XPO1, and KPT-350 administration increases the amount of endogenous IKB, thus inhibiting NFKB’s function.

Design/Methods:
In order to assess the short term effect of KPT-350 treatment on dystrophic disease phenotype and muscle architecture, sapje zebrafish (a severe model of DMD) embryos were treated from 1 to 5 days post-fertilization (dpf) with vehicle, 1.25 μM KPT-350, 2.5 μM KPT-350, or 2.5 μM aminophylline (positive control). In order to assess the effects of long-term KPT-350 treatment on survivability, sapje zebrafish were treated 3x/week for 24 hrs/dose with vehicle, 0.1 μM KPT-350, 1.0 μM KPT-350, or 2.5 μM aminophylline for 21 dpf.

Results:
In short-term treatment studies, KPT-350-treated sapje zebrafish showed significant prevention of the muscle degeneration pathology associated with dystrophin deficiency and improved overall muscle architecture as determined by histological analysis of myosin heavy chains. With long term treatment, KPT-350 extended the lifespan of the sapje zebrafish, with a significant number of KPT-350-treated sapje mutants surviving well past 10 dpf, and reduced overall dystrophic pathology.

Conclusions: KPT-350 is an IND-ready compound and our studies demonstrate that it can improve the symptoms associated with muscular dystrophy and is a promising small molecule compound for the treatment of dystrophindeficiency

b) MoveDMD Results: Effects of Edasalonexent, an NF-kB Inhibitor, in 4 to 7 Year Old Patients with Duchenne Muscular Dystrophy

Joanne Donovan1, Krista H. Elvire Vandenborne2, Lee Sweeney2, Gihan Tennekoon3, Erika Finanger4, Sabrina
Yum3, Perry Shieh5, Maria Mancini1, Pradeep Bista1, Andrew Nichols1, Richard Finkel6
1Catabasis Pharmaceuticals, 2University of Florida, 3The Children's Hospital of Philadelphia, 4Oregon Health
Science University, Child Neurology, 5UCLA, 6Nemours Children's Hospital

Objective: Assessment of safety and effects of edasalonexent (CAT-1004) on muscle in patients with Duchenne muscular dystrophy (DMD) using MRI.

Background: NF-κB is activated from infancy in DMD, driving inflammation, muscle degeneration and inhibiting muscle regeneration. Edasalonexent, an oral small molecule inhibiting NF-kB, demonstrated positive preclinical effects on skeletal, diaphragmatic and cardiac muscle in DMD models.

Design/Methods: MoveDMD is a Phase 1/2 three-part study in boys aged 4-7 with DMD not on glucocorticoids. Part A evaluated safety and pharmacokinetics (PK) of 3 doses of edasalonexent for 7 days (N=17), with exploratory measures of NF-κB activity. Additional patients (total N=31) were enrolled into a randomized, doubleblind, placebo-controlled 12-week dosing period with MRI T2 as the primary endpoint, and assessments including timed function tests, muscle strength, and North Star Ambulatory Assessment. A 36-week open-label extension
continues.

Results: As previously presented, during Part A, edasalonexent was generally well tolerated. AUC and Cmax were consistent with previously observed levels in adults at which NF-κB inhibition was seen. Genes enriched for NF-kB targets were assessed in whole blood by mRNA sequencing. Compared with baseline, two independently curated NF-kB gene-sets were significantly down-regulated. Several gene transcripts in the TLR and Fc receptor pathways, including TLR4 and FCGR2Awere reduced after a week on CAT-1004 (ANOVA p<0.05 and FDR<0.05
in pooled analysis compared to baseline).

Conclusions:
Safety, PK and NF-κB inhibition at edasalonexent doses of 67 and 100 mg/kg supported initiation of the 12-week, double-blind, placebo-controlled trial with T2 MRI of lower leg muscles as the primary endpoint, for which data will be presented. By reducing inflammation and muscle degeneration with potentially positive longer-term effects on muscle regeneration and function, Edasalonexent may have potential to be disease-modifying in DMD patients, regardless of mutation type.

c) Eteplirsen for the Treatment of Duchenne Muscular Dystrophy (DMD)

JS Charleston1, FJ Schnell1, J Dworzak1, C Donoghue1, J Lynch1, S Lewis2, L Chen2, LR Rodino-Klapac2, Z
Sahenk2, J Voss1, U DeAlwis1, DE Frank1, H Eliopoulos1, JR Mendell2
1Sarepta Therapeutics, Inc., 2Nationwide Children's Hospital

Objective:
Phosphorodiamidate morpholino oligomers (PMOs) are synthetic nucleic acid analogs that can be designed to sequence-specifically block spliceosomes from binding to dystrophin pre-mRNA, resulting in omission of the targeted exon from the transcript and restoration of the reading frame with the goal of enabling synthesis of internally-shortened dystrophin.

Background:
DMD, a rare, X-linked genetic disease results in progressive muscle degeneration and premature death. DMD is primarily caused by whole exon deletions in the dystrophin gene resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein.
Design/Methods: As of June 3, 2016, 81 of 150 treated patients had received weekly eteplirsen for ≥1 year.

Results:
PMO eteplirsen received accelerated approval in the US for patients with a dystrophin gene mutation amenable to exon 51 skipping based on an increase in dystrophin in skeletal muscle in some patients. Mean dystrophin increases as measured by Western blot were observed following 180 weeks of treatment in the pivotal Phase II Studies 201/202 when compared to untreated DMD controls (N=11; +0.85%, p=0.007) and at Week 48 in Phase III Study PROMOVI when compared to baseline (N=12; +0.28%, p=0.008). Immunohistochemistry analysis at
Week 180 in Study 201/202 also showed mean increases in dystrophin as measured by % dystrophin-positive fibers (N=11; +16.27%, p<0.001) and dystrophin immunofluorescence intensity (N=11; +13.20%, p<0.001) when compared to untreated DMD controls.
Adverse events occurring ≥25% more frequently than placebo in Study 201 were balance disorder, vomiting, and contact dermatitis. Serial echocardiograms conducted in Study 201/202 revealed no evidence of decline in left ventricular ejection fraction with >4.5 years of treatment.

Conclusions:
Eteplirsen is the first exon skipping therapy approved for the treatment of Duchenne muscular dystrophy amenable to exon 51 skipping. Lessons learned from the eteplirsen clinical development program can aid in development of PMO therapies targeting additional exons.

d) Eteplirsen, a Phosphorodiamidate Morpholino Oligomer (PMO) for Duchenne Muscular Dystrophy (DMD): Longitudinal Comparison to External Controls on Six-Minute Walk Test (6MWT) and Loss of Ambulation (LOA)

JR Mendell1, N Goemans2, LR Rodino-Klapac1, LP Lowes1, LN Alfano1, K Berry1, S Moody3, Emily Naughton4, E
Mercuri5, DMD Italian Network
1Nationwide Children's Hospital, 2University Hospitals Leuven, 3PharPoint Research, Inc., 4Sarepta Therapeutics,
Inc., 5Università Cattolica

Objective: PMO eteplirsen is designed to skip exon 51, restore the reading frame, and induce production of internally-shortened dystrophin in patients amenable to exon 51-skipping.

Background:
DMD, a rare, degenerative, X-linked disease occurring in ~1:5000 males worldwide results in progressive muscle loss and premature death. DMD is primarily caused by whole exon deletions resulting in a shift of the dystrophin mRNA reading frame that prevents production of functional dystrophin protein.

Design/Methods:
Analysis of 6MWT performance over 4 years compared boys treated with 30 or 50 mg/kg/wk eteplirsen IV (N=12) versus a group of comparable, untreated external controls (N=13) as defined by age, corticosteroid use, and genotype.

Results:
At Year 4, a statistically significant treatment benefit of 162 meters on 6MWT was observed in eteplirsen-treated patients versus external controls (p=0.0005). Sensitivity analyses of 6MWT with covariates including baseline 6MWT, age and glucocorticoid use resulted in differences >150 meters between the groups that were statistically significant (p<0.01).
Kaplan-Meier estimates of Loss of Ambulation (LOA) showed that 85% of the external control patients lost ambulation versus 17% of eteplirsen-treated patients at Year 4 (log-rank p=0.011). Median age to LOA in the external cohort was 12.9 years. The eteplirsen-treated patients had not reached median age of LOA as 10/12 were still ambulatory at Year 4; however, the median age of eteplirsen-treated patients at this time point was 13.4. No major safety signal was observed. Data from the final study time-point will be presented.

Conclusions: Eteplirsen slowed DMD progression as evidenced by a 162 meter advantage on the 6MWT compared to external control patients (p=0.0005) at Year 4. In addition, there was a reduction in risk of loss of ambulation in the eteplirsen treated patients (p=0.011). The 6MWT and LOA data reported here were not included in the US prescribing information for eteplirsen by the Food & Drug Administration.

e) Development of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Treatment of Duchenne Muscular Dystrophy (DMD)

P Duda1, GA Laforet1, C DiJohnson1, H Eliopoulos1, A Narayana1, DE Frank1, S Ruff1, JR Mendell2
1Sarepta Therapeutics, Inc., 2Nationwide Children's Hospital

Objective: PMOs are synthetic nucleotide analogs that can be designed to sequence-specifically block spliceosomes from binding to dystrophin pre-mRNA, resulting in omission of a deletion-adjacent exon from the transcript and restoration of the reading frame with the goal of enabling synthesis of internally-shortened dystrophin.

Background:
DMD, a rare, X-linked disease results in progressive muscle loss and premature death. DMD is primarily caused by whole exon deletions in the dystrophin gene resulting in an out-of-frame shift of the dystrophin mRNA reading frame that prevents production of functional dystrophin protein.

Design/Methods: Eteplirsen is a PMO that received accelerated approval in the US for patients with a DMD gene mutation amenable to exon 51 skipping. As of June 3, 2016, 150 patients aged 4-21 and amenable to exon 51 skipping have received eteplirsen in 7 clinical trials.

Results:
After 180 weeks of eteplirsen 30 or 50 mg/kg/wk IV (N=11 biopsied patients), some patients demonstrated increased dystrophin production by Western blot (mean increase +0.85%, p=0.007), % dystrophin-positive fibers (mean increase +16.27%, p<0.001), and immunofluorescence intensity (mean increase +13.20%, p<0.001) as compared to untreated DMD control samples. A statistically significant difference in eteplirsen-treated patients (N=12) in comparison to an untreated external control cohort (N=13) as measured by the 6-minute walk test was
shown after 4 years of follow-up (162m, p<0.001). No major safety signal was observed. SRP-4045 and SRP-4053 for DMD patients amenable to exon 45 or 53 skipping, respectively, are in clinical development A Phase III multi-national, randomized, double-blind, placebo-controlled trial to examine safety and efficacy of both SRP-4045 and -4053 is underway in the US. This will serve as the pivotal study for both PMOs and the foundation for the advancement of follow-on exon skipping programs.

Conclusions: PMOs have been shown to induce exon skipping and enable production of internally deleted dystrophin in some patients.

f) Reduced intramuscular blood flow correlates with more severe pathology in boys with
Duchenne/Becker muscular dystrophy

Alexander Dietz1, Anne Connolly1, Paul Golumbek1, Craig Zaidman1
1Washington University

Objective: To relate intramuscular blood flow to the degree of muscle pathology in two ways: by comparing Duchenne muscular dystrophy (DMD) to Becker muscular dystrophy (BMD) and by correlation with muscle echointensity.

Background: Intramuscular blood flow is thought to be reduced in D/BMD and may contribute to the pathology. Blood flow in small intramuscular vessels, unlike large vessels, cannot be quantified using color Doppler sonography. We use a previously developed protocol to quantify blood flow in small intramuscular vessels using power Doppler sonography in muscular dystrophy patients.

Design/Methods: We obtained ultrasound images of the anterior forearm (AF) and tibialis anterior (TA) muscles of boys with D/BMD. We quantified the change in intramuscular blood flow between rest and after one minute of exercise, measured as a percentage of muscle area. We compared the change in blood flow between boys with DMD, BMD, and historic controls, and correlate blood flow to the mean echointensity (grey scale level) measured from the same muscle.

Results: We imaged 8 boys with DMD and 3 boys with BMD ages 6-19 years old. Intramuscular blood flow (% median change (range)) in the AF was less in 7 of 8 forearms in DMD (0.25% (-0.47 – 2.19)) than the lowest BMD (2.46%(2.02 – 3.38), n=3) and the lowest historic control (2.16%). Intramuscular blood flow in the TA was lower in all (4/4) DMD (0.70% (0.16 – 1.89)) than the lowest BMD (5.08% (2.28 – 9.51), n=3) and the lowest historic control (3.91%). Intramuscular blood flow in boys with D/BMD decreased with higher muscle echogenicity (rs= -
0.6, p=0.007).

Conclusions: Intramuscular blood flow in boys with D/BMD can be quantified using power Doppler, and
reduction correlates with more severe pathology in DMD compared to BMD and as measured by echointensity

g) Awareness regarding respiratory care of DMD patients among child neurology providers: an international survey

Poonam Bhatia1, Rajeev Bhatia2
1Neurology, 2Pulmonology, Akron Children Hospital

Objective: To evaluate the awareness of respiratory care for Duchenne Muscular Dystrophy (DMD) patients among child neurology providers.

Background: Respiratory disease is the major cause of morbidity and mortality in DMD. Despite a 2004 American Thoracic Society (ATS) consensus statement on respiratory care of such patients, knowledge of appropriate management remains inadequate.

Design/Methods: The authors designed a 22-question survey (SurveyMonkey®) based on the 2004 American Thoracic Society guidelines(ATS) and distributed by email mainly to the global Child Neurology LISTSERV™ .

Results:
59 responded representing more than 30 centers around the world. 16% indicated they referred patients to a pulmonologist at the “onset of respiratory complaints” or “after confinement to a wheelchair”. This is inconsistent with ATS guidelines. 71% responded they order a chest X-ray in wheelchair-bound patients “only if clinically indicated” instead of an annual chest X-ray as per ATS guidelines. “Frequent clinical evaluation of scoliosis,” and “preoperative clearance by a pulmonologist prior to surgery” represented the most common
answers among respondents (93% and 90% respectively) which are consistent with the guidelines. Sleep-related breathing disorder symptoms were evaluated “frequently” at a clinic visit as per survey. Although the guidelines recommend an annual sleep study for wheelchair-bound patients, 52 % obtained these studies only when “clinically indicated.” Frequent swallow evaluation via history (as guidelines recommend) was the most common answer among respondents (88%). Regarding pulmonary management, 22% reported they were “unfamiliar”
with airway clearance techniques, although a majority was “familiar” or “somewhat familiar” with noninvasive (94%) and invasive (86%) mechanical ventilation.

Conclusions:
Overall, survey results showed consistency with most ATS guidelines. However, there remains insufficient awareness regarding some areas of respiratory care for DMD patients. Specifically, increased awareness regarding: the timing of referral to a pulmonologist, annual chest X-rays and sleep studies in wheelchairbound patients, can improve respiratory care and quality of life for these patients

h) Development and Implementation of Bone Health Protocol (BHP) in Duchenne Muscular Dystrophy (DMD)

Alexandra Otto1, Karen Loechner1, Sumit Verma1
1Emory/ Children's Healthcare of Altanta

Objective: To Develop/Implement BHP for DMD.

Background: Vitamin D (25OHD) deficiency, reduced bone mineral density (BMD) scores (dual-energy x-ray absorptiometry-DXA), and vertebral compression fractures (VFs) are common in DMD.

Design/Methods:
We retrospectively reviewed electronic medical records of 148 DMD boys for 25OHD, DXA scan frequency and scores, VFs and vitamin D therapies. Prospectively, use of the BHP in 2016 included obtaining 25OHD levels at 6 month intervals and DXA combined with spine radiographs (for those with spinal Z-score <-2.0 to assess for VFs) prior to initiation of steroids and, thereafter, annually. Intervention included treatment with vitamin D2 (ergocalciferol) 50,000 IU /week for 8 weeks for vitamin D deficiency/insufficiency and maintenance vitamin D3
(cholecalciferol) 1,000-2000 IU/day once therapeutic levels obtained. Dietary calcium supplementation augmented.

Results:
At baseline, 72% (107) DMD boys had one or more 25OHD levels available with 65% (70) having insufficient (<30 ng/ml) or deficient (<20 ng/ml) levels. 20% (30) boys had one or more DXA scans with 60% (18) having spine DXA Z-scores < -2.0 and 13% (4) having VFs. 25OHD deficiency/insufficiency correlated with low DXA Z scores. 63% (93) were on vitamin D supplementation [cholecalciferol 55% (51), calcitriol 45% (42) and ergocalciferol 6% (6)]. BHP implementation led to increased testing of 25OHD levels, DXA scans and spine films and more uniform treatment of 25OHD deficiency by providers (3 boys with deficient/insufficient 25OHD are now replete).

Conclusions: Although preliminary, we find that a BHP will, indeed, improve surveillance for vitamin D deficiency, decreased BMD and VFs in DMD boys. Using a uniform treatment approach we will assess effects of vitamin D/ dietary calcium supplementation on the above measures. The inclusion of bisphosphonate treatment in subpopulations of boys with DMD is anticipated.

i) Upper arm, pulmonary and cardiac magnetic resonance imaging for relative disease status in Duchenne muscular dystrophy

Ami Mankodi1, W.Patricia Bandettini2, William Kovacs3, Alexander Hanna2, Hirity Shimellis1, Chioma Nnamdi-
Emetarom1, Lasya Gaur2, Kenneth Fischbeck1, Jianhua Yao3, Andrew Arai2

1Neurogenetics Branch, NINDS, NIH, 2Advanced Cardiovascular Imaging, NHLBI, NIH, 3Radiology and Imaging
Sciences, NIH, Clinical Center

Objective:
To simultaneously assess the magnetic resonance imaging (MRI) biomarkers of disease activity in the upper arm and chest wall skeletal muscles, diaphragm, and myocardium of patients with Duchenne muscular dystrophy (DMD).

Background:
There is a need for biomarkers to assess disease activity and treatment response in DMD. Outcome measures targeting the lower limb function may not be applicable to patients who lose their independent mobility during the duration of the trial. Cardiomyopathy and restrictive lung disease cause early morbidity and mortality in patients with DMD.

Design/Methods:
We analyzed quantitative maps of T1 and T2 relaxation times and muscle fat fraction (FF) measurements in the MRI of the upper arm skeletal muscles and heart in ambulatory boys with DMD and age matched healthy volunteer boys. The chest wall and diaphragm motion and right lung area were assessed during deep breathing in the cineBreathing MRI in both groups.

Results:
The upper arm skeletal muscle FF and T2 values were increased in DMD boys than healthy volunteers. Myocardial FF was similar between the groups. Myocardial T1 and T2 values were shortened in DMD participants than healthy volunteers. The chest wall movement was reduced in DMD boys, whereas the diaphragm movement was similar to healthy volunteers. Right lung area was reduced during maximum inspiration and expiration in DMD boys compared to healthy volunteers.

Conclusions:
We detected fatty degeneration and edema in the upper arm skeletal muscles but not the myocardium in DMD boys who had normal ejection fraction. There was early involvement of the chest wall skeletal muscles but not diaphragm in DMD patients who had normal pulmonary function test. Imaging the heart and skeletal muscle using the same MRI methods may be useful in assessing relative disease status and therapeutic response in clinical trials of DMD.

j) Concentric exercise effects on skeletal muscle water T2 in Duchenne muscular dystrophy

Ami Mankodi1, Noura Azzabou3, Thomas Bulea4, Harmen Reyngoudt3, Hirity Shimellis1, Yupeng Ren5, Eunhee
Kim2, Kenneth Fischbeck1, Pierre Carlier3
1Neurogenetics Branch, 2Office of Biostatistics, NINDS, NIH, 3CEA, DRF, I²BM, MIRCen, NMR Laboratory,
Institute of Myology, 4Functional & Applied Biomechanics Section, Rehabilitation Medicine Department, NIH,
Clinical Center, 5RehabTek, Inc

Objective:
To examine the concentric ankle dorsiflexion exercise effects on the magnetic resonance imaging (MRI) measure of muscle water T2 in the lower legs of patients with Duchenne muscular dystrophy (DMD).

Background:
Surrogate outcome measures are needed to measure therapeutic response in clinical trials of DMD. Absence of dystrophin leads to sarcolemmal fragility with consequent more susceptibility to damage following eccentric exercise. Active movement training can lead to functional improvements. However, the effects of submaximal concentric muscle activity on DMD muscle are not yet known. MRI water T2 is sensitive to changes of muscle injury or edema, but fatty replacement masks increases in water T2 in human dystrophic muscle. The triexponential model measures muscle water T2 independent of fat values in skeletal muscle.

Design/Methods:
In 12 ambulatory DMD boys and 19 healthy volunteer boys, muscle water T2 was measured at baseline and 3h post concentric ankle dorsiflexion exercise in the lower leg muscles by the tri-exponential model. Muscle fat fraction (FF) was measured by Dixon.

Results:
The muscle FF was higher in nearly all of the lower leg muscles of DMD participants than the healthy volunteers (p < .001). At baseline muscle water T2 was higher in the lower leg muscles of DMD participants than the healthy volunteers (p < .001) with the exception of the extensor digitorum longus muscle. The DMD participants exerted significantly (p < .01) less power during exercise than healthy volunteers. Three hours after exercise, muscle water T2 increased from baseline in the lower legs of both groups. Most prominent T2 increases were in the ankle
dorsiflexor muscles of the DMD participants with a larger inter-subject variability than the healthy volunteers.

Conclusions: Skeletal muscle water T2 measured by the tri-exponential model is a sensitive MRI biomarker related
to muscle degeneration and concentric muscle exercise in DMD.

k) A Rare Case of Asymptomatic Becker Muscular Dystrophy


Fareed khan1, Usama Tariq, S.H Subramony, Alicia Parkar, Bilal Ansari
1Neurology, University of Florida

Objective: To describe an unusual case of asymptomatic Becker Muscular Dystrophy

Background: Becker muscular dystrophy is an X-linked recessive disorder characterized by slowly progressive muscle weakness, initially of the legs and pelvis and later of the cardiac muscles. This disease is caused by mutations in the dystrophin gene which encodes the protein dystrophin. Disease onset typically occurs by the third or fourth decades of life.

Design/Methods: A case report.

Results: A 61 year-old male was seen in clinic after genetic testing results. The patient's examination was without pathology. His creatine kinase level and an EKG were normal studies. The patient's seven year-old grandson had undergone a workup for muscle weakness and gait abnormality, for which he was found to have Becker Muscular Dystrophy. Both the patient and his grandson were found to have deletions in exons 48 and 49 in the dystrophin gene.

Conclusions:
In this study, we report a rare case of asymptomatic Becker Muscular Dsytrophy. Notably, while both the patient and his grandson had the same genetic mutation with deletions in exons 48 and 49, one of them was asymptomatic at 61 years old, while the other had an early onset at the age of 7. This report illustrates the need to further examine how the same mutation in the dystrophin gene may cause remarkably different phenotypes

l) A dedicated electrical impedance myography device in the assessment of Duchenne muscular dystrophy.

Craig Zaidman1, Seward Rutkove2, Julaine Florence1, Anne Connolly1, Brenda Wong3, Michele Yang4, Basil
Darras5, Kush Kapur5, Jose Bohorquez6
1Washington University, 2Neurology, Beth Israel Deaconess Medical Center/Harvard Medical School, 3Cincinnati
Children'S Hospital Medical Ctr, 4Children'S Hospital Colorado, 5Boston Children's Hospital/Harvard Medical
School, 6Skulpt Inc

Objective: To evaluate the sensitivity to disease progression of Duchenne muscular dystrophy (DMD) of a dedicated electrical impedance myography (EIM) device.

Background: Recent work in DMD has shown that EIM is sensitive to disease progression and the therapeutic benefit of corticosteroids. Most work to date has used custom-designed, off-the-shelf impedance devices and has been performed at a single center. Here we evaluate a dedicated EIM system that allows for rapid and improved data acquisition.

Design/Methods: In this 4-center study, healthy boys and those with DMD were assessed at 0, 3, 6 and 12 months after enrollment. The EIM 1103 System (Skulpt, Inc) was utilized with measurements performed on the dominant side on the deltoid, biceps brachii, wrist extensors, wrist flexors, vastus lateralis, tibialis anterior, and medial gastrocnemius. Multifrequency impedance data from 1kHz to 1MHz were collected on each muscle. Subjects in the 3-12-year-old group also underwent functional testing. A linear mixed effect model was applied, with random intercepts and slopes, alpha=0.05, 2-tailed for all analyses.

Results: Seventy-one boys with DMD and 72 healthy controls, mean age 8.2 years (range 0.6-17.4 years) and 8.3 years (range 0.3-17.9 years), respectively, were enrolled. A variety of multifrequency EIM measures demonstrated differences in the longitudinal EIM parameters over time in both younger and older boys. For example, the previously reported 100-500 kHz phase slope parameter averaged across all 7 muscles was
reduced (-0.0563 (standard error 0.0164)) in boys with DMD compared to controls (p=0.0009) at 12 months. Utilizing this measure as an outcome in a 12-month clinical trial would require a sample size of just 19 subjects/arm assuming an effect size of 1.0.

Conclusions: EIM multifrequency parameters obtained with this dedicated system are sensitive to disease progression in DMD. Further analysis of the EIM data and its relationship to the simultaneously obtained functional measures is underway.

m) Development of a validated Western blot method for quantification of human dystrophin protein used in Phase II and III clinical trials of eteplirsen for the treatment of Duchenne muscular dystrophy (DMD)

FJ Schnell1, C Donoghue1, J Dworzak1, JS Charleston1, DE Frank1, S Wilton2, S Lewis3, JR Mendell3, LR Rodino-
Klapac3, Z Sahenk3
1Sarepta Therapeutics, Inc., 2Murdoch University, 3Nationwide Children's Hospital

Objective:
Phosphorodiamidate morpholino oligomer (PMO) eteplirsen, which received accelerated approval in the US for patients with DMD gene mutations amenable to exon 51 skipping, along with other oligonucleotides under investigation in late-stage clinical trials, aim to enable production of internally-deleted dystrophin protein.

Background:
DMD is primarily caused by whole-exon deletions resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein. Western blotting has been traditionally used as the confirmatory assay for clinical diagnosis of DMD or Becker Muscular Dystrophy (BMD), but the methods are not standardized and lack true quantitative capacity.

Design/Methods:
A sensitive and semi-quantitative Western blot method was developed to detect dystrophin in human muscle biopsies utilizing a standard curve composed of non-DMD/BMD (normal control) muscle lysate spiked into DMD muscle lysate to maintain equivalent protein load. Running a 5-point standard curve ranging from 0.25% - 4% of a single normal control on every gel allows for calculation of dystrophin levels for unknown samples run on the same gel and enables normalization of slight fluctuations in dystrophin signal intensity between gels. Fixed film
exposure times ensure dystrophin signal is not oversaturated and remains within the dynamic range of the assay.

Results:
In an interim analysis following 48 weeks of treatment in a Phase III clinical study of eteplirsen, 12 biopsied patients showed a significant mean dystrophin increase by Western blot following treatment compared to their pre-treatment biopsies (P<0.05). This method allowed for confirmation of mean increases in dystrophin production following eteplirsen-treatment which were the basis of eteplirsen accelerated approval


Conclusions:
The Western blot method was validated according to FDA Draft Guidance for Industry, Bioanalytical Method Validation, and can provide confirmation of mechanism of action for dystrophin restoring therapies. The developed method enables sensitive and robust quantification of dystrophin down to 0.25% of “normal” levels.

FEBRUARY

19 - Left ventricular assist device as destination therapy in cardiac end-stage dystrophinopathies: Midterm results

 

JANUARY

15 - Myocardial Fibrosis Progression in Duchenne and Becker Muscular Dystrophy: A Randomized Clinical Trial