Elusive sources of variability of dystrophin rescue by exon skipping.

Volker Straub,Kanneboyina Nagaraju,Eric P. Hoffman,Alyson A. Fiorillo,Conner Shaughnessy,Marshall W. Hogarth,James S. Novak,Heather Gordish‐Dressman ,Qi Lü ,Kristy J. Brown,Yetrib Hathout,Margaret Benny Klimek,Maria Candida Vila,Sree Rayavarapu,John van den Anker,Umar Burki,Aiping Zhang,Kitipong Uaesoontrachoon,Terence A. Partridge,J Boehler

doi:10.1186/s13395-015-0070-6

Abstract

BackgroundSystemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Treatment with Phosphorodiamidate morpholino oligomers (PMO) lead to shorter de novo dystrophin protein in both animal models and DMD boys who otherwise lack dystrophin; however, restoration of dystrophin has been observed to be highly variable. Understanding the factors causing highly variable induction of dystrophin expression in pre-clinical models would likely lead to more effective means of exon skipping in both pre-clinical studies and human clinical trials.MethodsIn the present study, we investigated possible factors that might lead to the variable success of exon skipping using morpholino drugs in the mdx mouse model. We tested whether specific muscle groups or fiber types showed better success than others and also correlated residual PMO concentration in muscle with the amount of de novo dystrophin protein 1 month after a single high-dose morpholino injection (800 mg/kg). We compared the results from six muscle groups using three different methods of dystrophin quantification: immunostaining, immunoblotting, and mass spectrometry assays.ResultsThe triceps muscle showed the greatest degree of rescue (average 38±28 % by immunostaining). All three dystrophin detection methods were generally concordant for all muscles. We show that dystrophin rescue occurs in a sporadic patchy pattern with high geographic variability across muscle sections. We did not find a correlation between residual morpholino drug in muscle tissue and the degree of dystrophin expression.ConclusionsWhile we found some evidence of muscle group enhancement and successful rescue, our data also suggest that other yet-undefined factors may underlie the observed variability in the success of exon skipping. Our study highlights the challenges associated with quantifying dystrophin in clinical trials where a single small muscle biopsy is taken from a DMD patient.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0070-6) contains supplementary material, which is available to authorized users.

Highlights

Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle is a promising therapy
While the molecular basis for this variability is still unclear, we have recently described that muscle inflammation is linked to the production of TNF-alpha-induced microRNAs that target the dystrophin messenger RNA (mRNA) and could potentially influence the success of exon skipping in DMD [33]
Dystrophin rescue is highly variable after a single 800 mg/kg IV injection In order to fully characterize the level of dystrophin restored by exon skipping 1 month after a single highdose Phosphorodiamidate morpholino oligomers (PMO) injection in mdx mice, we analyzed six different muscles from six animals by immunofluorescent (IF) staining and immunoblotting (IB)

Summary

Introduction

Systemic delivery of anti-sense oligonucleotides to Duchenne muscular dystrophy (DMD) patients to induce de novo dystrophin protein expression in muscle (exon skipping) is a promising therapy. Vila et al Skeletal Muscle (2015) 5:44 expression using exon skipping with anti-sense oligonucleotides (AOs) in the muscle of patients [16,17,18,19,20,21,22,23] This approach partially repairs the patient’s dystrophin messenger (mRNA) by restoring the triplet codon reading frame, enabling translation of the patient’s RNA [20, 24, 25]. Human clinical trial data for exon skipping in DMD patients remain limited, but the few muscle biopsy data published far show highly variable dystrophin expression in patients’ muscle samples. Other studies to date have either not reported dystrophin rescue data [26] or the data were challenging to interpret [25, 27]

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Discussion

Conclusion