Combination Treatment of Erythromycin and Furamidine Provides Additive and Synergistic Rescue of Mis-splicing in Myotonic Dystrophy Type 1 Models

Abstract

Myotonic dystrophy type 1 (DM1) is a multi-systemic disease that presents with clinical symptoms including myotonia, cardiac dysfunction and cognitive impairment. DM1 is caused by a CTG expansion in the 3’ UTR of the DMPK gene. The transcribed expanded CUG repeat RNA sequester the muscleblind-like (MBNL) and up-regulate the CUG-BP Elav-like (CELF) families of RNA-binding proteins leading to global mis-regulation of RNA processing and altered gene expression. Currently, there are no disease-targeting treatments for DM1. Given the multi-step pathogenic mechanism, combination therapies targeting different aspects of the disease mechanism may be a viable therapeutic approach. Here, as proof-of-concept, we studied a combination of two previously characterized small molecules, erythromycin and furamidine, in two DM1 models. In DM1 patient-derived myotubes, rescue of mis-splicing was observed with little to no cell toxicity. In a DM1 mouse model, a combination of erythromycin and the prodrug of furamidine (pafuramidine), administered orally, displayed both additive and synergistic mis-splicing rescue. Gene expression was only modestly affected and over 40 % of the genes showing significant expression changes were rescued back toward WT expression levels. Further, the combination treatment partially rescued the myotonia phenotype in the DM1 mouse. This combination treatment showed a high degree of mis-splicing rescue coupled with low off-target gene expression changes. These results indicate that combination therapies are a promising therapeutic approach for DM1.

A CTG repeat-selective chemical screen identifies microtubule inhibitors as selective modulators of toxic CUG RNA levels

“…Immunofluorescence Microscopy. The protocol was carried out as described with minor modifications (66). Frozen vastus lateralis (quadriceps) muscle from HSA LR mice was sectioned into 8-μm slices onto slides and fixed with 4% PFA for 15 min at room temperature (RT), permeabilized by using 1:1 methanol:acetone (prechilled) for 5 min at −20°C, and blocked by using Background Sniper for at least 30 min at RT (Biocare Medical).…”

“…Toxicity Analysis in Cell Culture. The protocol was carried out as described with minor modifications (66). For myoblasts, ∼1 × 10 4 myoblasts were plated per well in 96-well plates in SkGM-2 BulletKit growth medium (Lonza).…”

“…RNA-Seq Library Preparation. The protocol was carried out as described with minor modifications (66). RNA was isolated from cells by using an Aurum Total RNA mini kit (Bio-Rad) according to the package insert with on-column DNase1 treatment.…”

“…Transcriptome Analysis from RNA-Seq Data. The protocol was carried out as described with minor modifications (66). Raw reads were checked for quality and aligned to GRCm38.p5 mouse genome by using STAR (Version 2.5.1b) (67) and a .gtf file generated from Version M16 GENECODE gene models.…”

Mitigating RNA Toxicity in Myotonic Dystrophy using Small Molecules

“…As proof of concept, a recent study reported that two previously characterized compounds that separately displayed efficacy in DM1 models, furamidine and erythromycin, displayed an even greater rescue of mis-splicing in combination than expected from a mere additive effect [110]. Importantly, this combination treatment yielded lower toxicity and fewer off-target effects than when either drug was administered alone in DM1 patient and mouse models [110]. Hence, if combination treatments are a viable therapeutic strategy for treating DM, there are already many unexplored therapeutic avenues that could potentially hold promise.…”