P.145Optimisation of a high–throughput digital script for multiplexed immunofluorescent analysis of sarcolemmal dystrophin - associated protein complex (DPC) and myofibre regeneration in entire transverse sections of muscle biopsies in Duchenne muscular dystrophy

ObjectiveTo report safety, pharmacokinetics, exon 53 skipping, and dystrophin expression in golodirsen-treated patients with Duchenne muscular dystrophy (DMD) amenable to exon 53 skipping.MethodsPart 1 was a randomized, double-blind, placebo-controlled, 12-week dose titration of once-weekly golodirsen; part 2 is an ongoing, open-label evaluation. Safety and pharmacokinetics were primary and secondary objectives of part 1. Primary biological outcome measures of part 2 were blinded exon skipping and dystrophin protein production on muscle biopsies (baseline, week 48) evaluated, respectively, using reverse transcription PCR and Western blot and immunohistochemistry.ResultsTwelve patients were randomized to receive golodirsen (n = 8) or placebo (n = 4) in part 1. All from part 1 plus 13 additional patients received 30 mg/kg golodirsen in part 2. Safety findings were consistent with those previously observed in pediatric patients with DMD. Most of the study drug was excreted within 4 hours following administration. A significant increase in exon 53 skipping was associated with ∼16-fold increase over baseline in dystrophin protein expression at week 48, with a mean percent normal dystrophin protein standard of 1.019% (range, 0.09%–4.30%). Sarcolemmal localization of dystrophin was demonstrated by significantly increased dystrophin-positive fibers (week 48, p < 0.001) and a positive correlation (Spearman r = 0.663; p < 0.001) with dystrophin protein change from baseline, measured by Western blot and immunohistochemistry.ConclusionGolodirsen was well-tolerated; muscle biopsies from golodirsen-treated patients showed increased exon 53 skipping, dystrophin production, and correct dystrophin sarcolemmal localization.Clinicaltrials.gov identifierNCT02310906.Classification of evidenceThis study provides Class I evidence that golodirsen is safe and Class IV evidence that it induces exon skipping and novel dystrophin as confirmed by 3 different assays.

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Development of digital tissue image analysis solution for muscle biopsies in support of disease-modifying therapies for Duchenne muscular dystrophy

Neuromuscular Disorders ◽

10.1016/j.nmd.2016.06.253 ◽

2016 ◽

Vol 26 ◽

pp. S155 ◽

Cited By ~ 1

Author(s):

J. Tinsley ◽

C. Pulliam ◽

A. Milici ◽

F. Aeffner ◽

K. Davies ◽

...

Keyword(s):

Image Analysis ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Disease Modifying Therapies ◽

Disease Modifying ◽

Muscle Biopsies

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Function and Genetics of Dystrophin and Dystrophin-Related Proteins in Muscle

Physiological Reviews ◽

10.1152/physrev.00028.2001 ◽

2002 ◽

Vol 82 (2) ◽

pp. 291-329 ◽

Cited By ~ 721

Author(s):

Derek J. Blake ◽

Andrew Weir ◽

Sarah E. Newey ◽

Kay E. Davies

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Protein Complex ◽

Muscle Disease ◽

Calcium Handling ◽

Dystrophic Muscle ◽

Gene Encoding ◽

Muscle Wasting Disease ◽

Novel Therapeutic Strategies ◽

Related Proteins

The X-linked muscle-wasting disease Duchenne muscular dystrophy is caused by mutations in the gene encoding dystrophin. There is currently no effective treatment for the disease; however, the complex molecular pathology of this disorder is now being unravelled. Dystrophin is located at the muscle sarcolemma in a membrane-spanning protein complex that connects the cytoskeleton to the basal lamina. Mutations in many components of the dystrophin protein complex cause other forms of autosomally inherited muscular dystrophy, indicating the importance of this complex in normal muscle function. Although the precise function of dystrophin is unknown, the lack of protein causes membrane destabilization and the activation of multiple pathophysiological processes, many of which converge on alterations in intracellular calcium handling. Dystrophin is also the prototype of a family of dystrophin-related proteins, many of which are found in muscle. This family includes utrophin and α-dystrobrevin, which are involved in the maintenance of the neuromuscular junction architecture and in muscle homeostasis. New insights into the pathophysiology of dystrophic muscle, the identification of compensating proteins, and the discovery of new binding partners are paving the way for novel therapeutic strategies to treat this fatal muscle disease. This review discusses the role of the dystrophin complex and protein family in muscle and describes the physiological processes that are affected in Duchenne muscular dystrophy.

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P1.6 The incidence of revertant and trace dystrophin expression in muscle biopsies of Duchenne Muscular Dystrophy patients with different exon deletions

Neuromuscular Disorders ◽

10.1016/j.nmd.2011.06.766 ◽

2011 ◽

Vol 21 (9-10) ◽

pp. 643 ◽

Cited By ~ 6

Author(s):

A. Lourbakos ◽

J. Sipkens ◽

C. Beekman ◽

D. Kreuger ◽

L. Brasz ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Dystrophin Expression ◽

Muscle Biopsies

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Mononuclear cell analysis of muscle biopsies in prednisone- and azathioprine-treated Duchenne muscular dystrophy

Neurology ◽

10.1212/wnl.43.3_part_1.532 ◽

1993 ◽

Vol 43 (3, Part 1) ◽

pp. 532-532 ◽

Cited By ~ 45

Author(s):

J. T. Kissel ◽

D. J. Lynn ◽

K. W. Rammohan ◽

J. P. Klein ◽

R. C. Griggs ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mononuclear Cell ◽

Cell Analysis ◽

Muscle Biopsies

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High Throughput Screening in Duchenne Muscular Dystrophy: From Drug Discovery to Functional Genomics

Biology ◽

10.3390/biology3040752 ◽

2014 ◽

Vol 3 (4) ◽

pp. 752-780 ◽

Cited By ~ 7

Author(s):

Thomas Gintjee ◽

Alvin Magh ◽

Carmen Bertoni

Keyword(s):

Drug Discovery ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Functional Genomics ◽

High Throughput ◽

High Throughput Screening

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Clinical and Experimental Results on Cardiac Troponin Expression in Duchenne Muscular Dystrophy

Clinical Chemistry ◽

10.1093/clinchem/47.3.451 ◽

2001 ◽

Vol 47 (3) ◽

pp. 451-458 ◽

Cited By ~ 31

Author(s):

Angelika Hammerer-Lercher ◽

Petra Erlacher ◽

Reginald Bittner ◽

Rudolf Korinthenberg ◽

Daniela Skladal ◽

...

Keyword(s):

Skeletal Muscle ◽

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Western Blot ◽

Blot Analysis ◽

Cardiac Troponin ◽

Western Blot Analysis ◽

Clinical Evidence ◽

Mdx Mouse ◽

Muscle Biopsies

Abstract Background: Because of controversial earlier studies, the purpose of this study was to provide novel experimental and additional clinical data regarding the possible reexpression of cardiac troponin T (cTnT) in regenerating skeletal muscle in Duchenne muscular dystrophy (DMD). Methods: Plasma from 14 patients (mean age, 7.5 years; range, 5.7–19.4 years) with DMD was investigated for creatine kinase (CK), the CK MB isoenzyme (CKMB), cTnT and cardiac troponin I (cTnI), and myoglobin. cTnT concentrations were measured by an ELISA (second-generation assay; Roche) using the ES 300 Analyzer. cTnI, myoglobin, and CKMB were measured by an ELISA using the ACCESS System (Beckman Diagnostics). Troponin isoform expression was studied by Western blot analysis in remnants of skeletal muscle biopsies of three patients with DMD and in an animal model of DMD (mdx mice; n = 6). Results: There was no relation of cTnT and cTnI to clinical evidence for cardiac failure. cTnI concentrations remained below the upper reference limit in all patients. cTnT was increased (median, 0.11 μg/L; range, 0.06–0.16 μg/L) in 50% of patients. The only significant correlation was found for CK (median, 3938 U/L; range, 2763–5030 U/L) with age (median, 7.5 years; range, 6.8–10.9 years; r = −0.762; P = 0.042). Western blot analysis of human or mouse homogenized muscle specimens showed no evidence for cardiac TnT and cTnI expression, despite strong signals for skeletal muscle troponin isoforms. Conclusions: We found no evidence for cTnT reexpression in human early-stage DMD and in mdx mouse skeletal muscle biopsies. Discrepancies of cTnT and cTnI in plasma samples of DMD patients were found, but neither cTnT nor cTnI plasma concentrations were related with other clinical evidence for cardiac involvement.

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