Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease

Abstract

Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

Keywords: Duchenne Muscular Dystrophy; Spinal Muscular Atrophy; antisense oligonucleotides; clinical trials; oligonucleotides delivery.

Figure 1

Chemical modifications of ASO backbone. PS, phosphorotioate; NP, N3′-P5′ phosphoroamidate; OMe, 2′-O-methyl; MOE, 2′-O-methoxy-ethyl; LNA, locked nucleic acid; PMO, phosphoroamidate morpholino; PNA, peptide nucleic acid; tcDNA, tricyclo DNA.

Figure 2

Antisense-mediated exon skipping mechanisms. (a) Cryptic splicing mutations induce the inclusion of an aberrant exon (black box) into the mature transcript and the ASO can skip the cryptic exon restoring the normal transcript; (b) exon inclusion can be induced by ASO that, targeting the intronic splicing silencers (ISS) generated by the mutation, restores the correct exon inclusion (red box); (c) ASO can be used for switching between alternative splicing isoforms; (d) ASO can support exon skipping restoring the correct reading frame; (e) ASO can induce the translation of an internally deleted protein or can generate reading frame disruptions with consequent partially or complete transcript knockdown.

Figure 3

DMD antisense oligonucleotides in clinical trials. Data extrapolated from https://clinicaltrials.gov February 2017.

Figure 4

Schematic representation of SMN2 exon 7 splicing regulatory elements and ASO targets. The functional elements regulating exon 7 splicing in SMN2 are depicted at the top of the figure. ASOs targeting these regulatory elements are listed below, including bi-functional ASO (grey oval) and conventional ASOs (yellow rectangle).