Link between MHC Fiber Type and Restoration of Dystrophin Expression and Key Components of the DAPC by Tricyclo-DNA-Mediated Exon Skipping.

Saleh Omairi,Luis Garcia,Federica Montanaro,Kwan-Leong Hau,Ketan Patel,Henry Collin-Hooper,Aurelie Goyenvalle

doi:10.1016/j.omtn.2017.10.014

Abstract

Exon skipping mediated by tricyclo-DNA (tc-DNA) antisense oligonucleotides has been shown to induce significant levels of dystrophin restoration in mdx, a mouse model of Duchenne muscular dystrophy. This translates into significant improvement in key disease indicators in muscle, cardio-respiratory function, heart, and the CNS. Here we examine the relationship between muscle fiber type, based on myosin heavy chain (MHC) profile, and the ability of tc-DNA to restore not only dystrophin but also other members of the dystrophin-associated glycoprotein complex (DAPC). We first profiled this relationship in untreated mdx muscle, and we found that all fiber types support reversion events to a dystrophin-positive state, in an unbiased manner. Importantly, we show that only a small fraction of revertant fibers expressed other members of the DAPC. Immunoblot analysis of protein levels, however, revealed robust expression of these components, which failed to correctly localize to the sarcolemma. We then show that tc-DNA treatment leads to nearly all fibers expressing not only dystrophin but also other key components of the DAPC. Of significance, our work shows that MHC fiber type does not bias the expression of any of these important proteins. This work also highlights that the improved muscle physiology following tc-DNA treatment reported previously results from the complete restoration of the dystrophin complex in all MHCII fibers with equal efficiencies.

Highlights

Duchenne muscular dystrophy (DMD) affects 1:5,000 male births, and it is the most common fatal childhood muscular disease.[1,2] Mutations in the DMD gene affect expression of dystrophin, a protein normally localized to the inner surface of the sarcolemma in muscle fibers.[3,4] Dystrophin together with a number of other proteins that constitute the dystrophin-associated glycoprotein complex (DAPC) acts to link the muscle fiber cytoskeleton, the sarcolemma, and the extracellular matrix (ECM) into a functional unit that maintains muscle integrity.[5,6] The DAPC is composed of three sub-complexes: (1) the sarcoglycans (a, b, g, and d); (2) syntrophin, nNOS, and dystrobrevin; and (3) a and b dystroglycan
We investigated the relationship between muscle compositions in terms of myosin heavy chain (MHC) fiber type and dystrophin restoration by tc-DNA antisense oligonucleotides (AONs) with a view to developing an understanding of its specificity of action
We commenced the study by comparing the MHC profile of the tibialis anterior (TA) muscle in the three cohorts under investigation: WT, mdx, and tc-DNA-treated mdx mice

Summary

Introduction

Duchenne muscular dystrophy (DMD) affects 1:5,000 male births, and it is the most common fatal childhood muscular disease.[1,2] Mutations in the DMD gene affect expression of dystrophin, a protein normally localized to the inner surface of the sarcolemma in muscle fibers.[3,4] Dystrophin together with a number of other proteins that constitute the dystrophin-associated glycoprotein complex (DAPC) acts to link the muscle fiber cytoskeleton, the sarcolemma, and the extracellular matrix (ECM) into a functional unit that maintains muscle integrity.[5,6] The DAPC is composed of three sub-complexes: (1) the sarcoglycans (a, b, g, and d); (2) syntrophin, nNOS, and dystrobrevin; and (3) a and b dystroglycan. Restoration of dystrophin expression by exon skipping has been proven to be efficacious in vitro, in animal models and in DMD patients.[11,12,13] Several classes of chemical modifications have been developed for AON-mediated exon skipping, among which are 20O-methylribooligonucleosidephosphorothioate (20OMe), phosphorodiamidate morpholino oligomers (PMOs), and tricyclo-DNA (tc-DNA) The latter has a number of properties that make it an attractive chemistry to exploit for therapeutic uses, including high RNA affinity, resistance to nuclease activity, and the ability to form nanoparticles that may facilitate uptake into cells.[14,15,16] We have recently shown, using mdx mice as a rodent model for DMD, that tc-DNA mediates unprecedented levels of exon skipping after systemic delivery in skeletal muscle and in the heart and brain.[16] This translated into normalization of specific force in the tibialis anterior muscle as well as improved cardiovascular function and the correction of behavioral characteristics.[16]

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