Gapmer Antisense Oligonucleotides Suppress the Mutant Allele of COL6A3 and Restore Functional Protein in Ullrich Muscular Dystrophy.

Elena Marrosu,Haiyan Zhou,Pierpaolo Ala,Francesco Muntoni

doi:10.1016/j.omtn.2017.07.006

Elena Marrosu, Haiyan Zhou + Show 2 more

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Dominant-negative mutations in the genes that encode the three major α chains of collagen type VI, COL6A1, COL6A2, and COL6A3, account for more than 50% of Ullrich congenital muscular dystrophy patients and nearly all Bethlem myopathy patients. Gapmer antisense oligonucleotides (AONs) are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In this study, we exploited the application of the allele-specific silencing approach by gapmer AON as a potential therapy for Collagen-VI-related congenital muscular dystrophy (COL6-CMD). A series of AONs were designed to selectively target an 18-nt heterozygous genomic deletion in exon 15 of COL6A3 at the mRNA and pre-mRNA level. We showed that gapmer AONs can selectively suppress the expression of mutant transcripts at both pre-mRNA and mRNA levels, and that the latter strategy had a far stronger efficiency than the former. More importantly, we found that silencing of the mutant transcripts by gapmer AONs increased the deposition of collagen VI protein into the extracellular matrix, thus restoring functional protein production. Our findings provide a clear proof of concept for AON allele-specific silencing as a therapeutic approach for COL6-CMD.

Highlights

Collagen-VI-related congenital muscular dystrophies (COL6-CMD) are the second most common diagnosis in congenital muscular dystrophies according to a recent retrospective review performed by our center on genetic studies in childhood neuromuscular diseases in the UK population.[1]
The specific enzyme recruited by the antisense oligonucleotides (AONs)-RNA duplex is RNase H1.19 It has been reported that RNase H active AONs exert their action predominantly in the nucleus, where they interact with the pre-mRNA20, whereas in other reports, mRNA in the cytoplasm is the predominant target of RNase H active AONs.[18]
We followed the classic design of gapmer AON: the RNase H activating phosphorothioate DNA domain (DNA gap) is flanked by short RNA sequences in the 20-OMe backbone at both ends to protect the AONs from degradation by nucleases.[22]

Summary

Introduction

Collagen-VI-related congenital muscular dystrophies (COL6-CMD) are the second most common diagnosis in congenital muscular dystrophies according to a recent retrospective review performed by our center on genetic studies in childhood neuromuscular diseases in the UK population.[1]. The assembly begins with the formation of the basic monomer, which is composed of one of each of the three a chain subunits encoded by the three collagen 6 genes. Within the cell, these monomers associate to form dimers, which pair up into tetramers. These monomers associate to form dimers, which pair up into tetramers These tetramers are secreted into the ECM, where they assemble in an end-to-end fashion to give rise to the final microfibrillar network.[5,6] Mutations in any of the three collagen 6 genes disrupt the correct formation of tetramers and they fail to be secreted into the ECM to form collagen VI microfibrillar structures (Figure 1).[3] The role of collagen VI in muscle is not completely understood. A more recent study in organizing the satellite cell niche, which regulates muscle regeneration, was demonstrated.[8]

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