Free Muscleblind (MBNL) protein levels are thought to be the linchpin in the pathogenesis of myotonic dystrophy. Sequestration of MBNL by binding to hairpin structures formed expanded DMPK RNA repeats leads to missplicing of mRNAs essential to the normal structure and function of the various tissues impacted by DM. Thus preservation of functionally significant intracellular levels of MBNL represents a viable strategy for therapy development. A new publication by Beatriz Llamusi, Ruben Artero, and colleagues (University of Valencia, Incliva Health Research Institute, and Joint Unit Incliva-CIPF) identifies mIR regulators of MBNL as potential therapy development targets for DM1 (Cerro-Herreros et al., 2018).

Identifying miR Suppressors of Mammalian MBNL

Multiple lines of evidence have established MBNL up-regulation as a potentially effective target for novel therapies in DM1. A prior study in a DM1 Drosophila model (Cerro-Herreros et al., 2016) showed reversal of missplicing events, reduced skeletal muscle pathology, improved muscle function, and increased lifespan via suppression of two micro RNAs (miRs) known to negatively regulate MBNL. A new publication from this same team extends these findings to mammalian cell and animal model systems.

Based on their prior work in the fly, the research team overexpressed a panel of miRs in HeLa cells, identifying those that reduced MBNL1 or MBNL2 transcript levels by a threshold of 4-fold or higher. miRs were selected for further analysis based on the HeLa assay data and in silico predictions of MBNL binding strength. Additional studies confirmed a miR subset (miR-96, miR-218, and miR-23b) as binding to the MBNL 3’-UTR and negatively impacting both MBNL transcript and protein levels.

Antagonizing miR-23b and miR-218 in vitro Stabilizes MBNL mRNA

Two of the miRs found to bind and suppress MBNL, miR-23b and miR-218, were also highly expressed in DM1myoblasts and muscle biopsies. Targeting DM1 myoblasts with antisense oligonucleotide (“antagomiR”) to miR-218 showed substantial (50% > controls) and dose-dependent increases in MBNL transcripts. By contrast, miR-23b anatagomiR did not show classic dose-response behavior, but was effective at a lower dose and exhibited clear mechanism of action in directly reducing miR-23b levels and thereby increasing MBNL transcripts. Preliminary toxicology studies suggested that the two antagomiRs were effective at concentrations below toxicity threshold.

Antagonizing miR-23b and miR-218 Rescues Molecular, Structural, and Functional Consequences of DM1

In a series of studies, the research team developed evidence to support antagonism of miR-23b and miR-218 as potentially viable paths toward a therapeutic for DM1. This evidence included: both antagomiRs were shown to rescue splicing defects in DM1 patient-derived cells; both restored normal cellular localization of MBNL protein; both increased Mbnl expression in HSA^LR mouse skeletal muscle; both rescued multiple splicing events in HSA^LR mice; and both improved skeletal muscle histopathology and reduced myotonia in HSA^LR mice. In additional studies, the research team showed continued miR target reduction and molecular, structural (skeletal muscle central nuclei), and functional (forelimb grip and myotonia) efficacy at 6 weeks after antagomiR injection.

Synthesis

This publication makes a compelling case that targeting the suppressive activity of miR-23b and miR-218 to up-regulate MBNL is a potential therapeutic strategy for DM1. Both miRs were found to be expressed in organ systems impacted by DM1 (skeletal muscle, heart, and CNS were assessed) and suppression of either modulated the molecular, structural, and/or functional consequences of DM1 in appropriate cell or animal models. There are several considerations before this target validation work can translate into the clinic, including, but not limited to, performance of a definitive proof of concept study (independent replication under rigorous conditions is essential for any therapeutic development effort) and either optimization of the currently limited intracellular delivery capacity of antisense oligonucleotides or development of small molecule inhibitors of miR-23b or miR-218.

References:

Derepressing muscleblind expression by miRNA sponges ameliorates myotonic dystrophy-like phenotypes in Drosophila.
Cerro-Herreros E, Fernandez-Costa JM, Sabater-Arcis M, Llamusi B, Artero R.
Sci Rep. 2016 Nov 2;6:36230. doi: 10.1038/srep36230.

miR-23b and miR-218 silencing increase Muscleblind-like expression and alleviate myotonic dystrophy phenotypes in mammalian models.
Cerro-Herreros E, Sabater-Arcis M, Fernandez-Costa JM, Moreno N, Perez-Alonso M, Llamusi B, Artero R.
Nat Commun. 2018 Jun 26;9(1):2482. doi: 10.1038/s41467-018-04892-4.