Identification of serum protein biomarkers for utrophin based DMD therapy

Abstract

Despite promising therapeutic avenues, there is currently no effective treatment for Duchenne muscular dystrophy (DMD), a lethal monogenic disorder caused by the loss of the large cytoskeletal protein, dystrophin. A highly promising approach to therapy, applicable to all DMD patients irrespective to their genetic defect, is to modulate utrophin, a functional paralogue of dystrophin, able to compensate for the primary defects of DMD restoring sarcolemmal stability. One of the major difficulties in assessing the effectiveness of therapeutic strategies is to define appropriate outcome measures. In the present study, we utilised an aptamer based proteomics approach to profile 1,310 proteins in plasma of wild-type, mdx and Fiona (mdx overexpressing utrophin) mice. Comparison of the C57 and mdx sera revealed 83 proteins with statistically significant >2 fold changes in dystrophic serum abundance. A large majority of previously described biomarkers (ANP32B, THBS4, CAMK2A/B/D, CYCS, CAPNI) were normalised towards wild-type levels in Fiona animals. This work also identified potential mdx markers specific to increased utrophin (DUS3, TPI1) and highlights novel mdx biomarkers (GITR, MYBPC1, HSP60, SIRT2, SMAD3, CNTN1). We define a panel of putative protein mdx biomarkers to evaluate utrophin based strategies which may help to accelerate their translation to the clinic.Duchenne muscular dystrophy (DMD) is a lethal X-linked recessive disorder caused by mutations in the dystrophin gene 1 . This disorder affects 1 in 5000 boys 2 and is characterized by a progressive muscle wasting leading to loss of ambulation by 8-12 years of age 3 and death by early adulthood due to cardiorespiratory failure 4 . Dystrophin, an essential link between the dystrophin associated protein complex (DAPC) at the sarcolemma and the cytoskeleton, maintains the strength, flexibility and stability in skeletal muscles 5 . In the absence of dystrophin, the myofibres are more susceptible to contraction-induced injury which results in muscle wasting and premature death 6 . There is currently no effective treatment for the disease. Glucocorticoid treatment is the current standard of care which delays the loss of ambulation by 3-4 years 7,8 but shows no long treatment benefit and is often associated with debilitating side effects [9][10][11] . The urgency to seek a therapy for DMD has resulted in parallel efforts to develop exon skipping 12,13 , termination codon read through 14 , dystrophin gene replacement or editing therapies 15,16 and non-dystrophin strategies [17][18][19] such as utrophin modulation 20,21 . However, despite the recent accelerated approval of Exondys 51 (eteplirsen) in US, disappointing clinical trials results 22 and failure of approval from the FDA for Ataluren 23 and Kyndrisa 24 drugs rekindle discussions about clinical trials designs and endpoints. We have focused on utrophin modulation because it is applicable to all DMD patients irrespective of their dystrophin mutation. Utrophin is found at the sarcolemma in utero and is progressive...

The SINE Compound KPT-350 Blocks Dystrophic Pathologies in DMD Zebrafish and Mice

“…Serum cytokines are a valid biomarker for dystrophin-deficient skeletal muscle in dystrophic mice, dogs, and human patient samples. [54][55][56] We analyzed whole serum taken from each of the four experimental cohorts via a mouse cytokine array panel to determine whether known biomarkers of dystrophin deficiency were altered in the KPT-350treated D2-mdx mice. Vehicle-treated D2-mdx mice showed a significant upregulation of key pro-inflammatory and apoptosisrelated cytokines, such as TNF-a, interferon gamma (IFNg), chemokine (C-X-C motif) ligand 16 (CXCL16), osteopontin (Spp1), IL-1a, IL-1b, IL-2, IL-6, and CD95L, compared with vehicle-treated WT control serum ( Figures 5A-5I).…”

Assessment of Variability in the SOMAscan Assay

“…Due to evolutionary conservation, SOMAscan has proven useful in some applications to non-human species as well, as shown e.g. in recent mouse studies 12 , 13 .…”

Insights into the Pathogenic Secondary Symptoms Caused by the Primary Loss of Dystrophin

“…Affected boys exhibit symptoms between 2 and 5 years of age, including motor developmental delay, abnormal gait, and muscle weakness [1]. The progressive muscle degeneration leads to loss of ambulation at 8-12 years with premature death due to cardio-respiratory failure [2]. The genetic causes of the disease are "out of frame" deletions (68%), duplications (11%), and small mutations (20%) in the dystrophin gene, the largest known gene with the highest spontaneous mutation rates in humans [3,4].…”

“…The perturbation of calcium homeostasis along with chronic inflammation, mitochondrial dysfunction, and elevated oxidative stress characterize the dystrophic muscle and milieu. Moreover, repeated cycles of myofiber necrosis and ineffective regeneration contribute to the establishment of a chronic inflammatory response, which in turn amplifies tissue damage, leading to muscle degeneration and fatty tissue replacement [2,9]. Because no etiologic therapy is available for Duchenne muscular dystrophy, a better understanding of secondary pathogenic mechanisms downstream from the primary defect could be useful in developing new adjuvant treatments.…”