Allele-specific silencing therapy for Dynamin 2-related dominant centronuclear myopathy.

Delphine Trochet,Maud Beuvin,Cécile Peccate,Aymen Rabai,Marc Bitoun,Jocelyn Laporte,Stéphanie Lorain,Stéphane Vassilopoulos,Arnaud Ferry,Sofia Benkhelifa‐Ziyyat,Kamel Mamchaoui,Pascale Guicheney,Laura Julien,Bernard Prudhon

doi:10.15252/emmm.201707988

Abstract

Rapid advances in allele‐specific silencing by RNA interference established a strategy of choice to cure dominant inherited diseases by targeting mutant alleles. We used this strategy for autosomal‐dominant centronuclear myopathy (CNM), a rare neuromuscular disorder without available treatment due to heterozygous mutations in the DNM2 gene encoding Dynamin 2. Allele‐specific siRNA sequences were developed in order to specifically knock down the human and murine DNM2‐mRNA harbouring the p.R465W mutation without affecting the wild‐type allele. Functional restoration was achieved in muscle from a knock‐in mouse model and in patient‐derived fibroblasts, both expressing the most frequently encountered mutation in patients. Restoring either muscle force in a CNM mouse model or DNM2 function in patient‐derived cells is an essential breakthrough towards future gene‐based therapy for dominant centronuclear myopathy.

Highlights

Autosomal-dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy without available curative treatment and with pathomechanisms still largely unknown
A screening for allele-specific siRNAs capable of silencing the mutant Dnm2 allele without affecting the WT allele was performed in mouse embryonic fibroblasts (MEFs) cultured from heterozygous (HTZ) KI-Dnm2 embryos
This property qualified allele-specific RNAi to target single nucleotide substitutions representing the majority of the 26 Dynamin 2 (DNM2) mutations identified in AD-CNM, especially for the

Summary

Introduction

Autosomal-dominant centronuclear myopathy (AD-CNM) is a rare congenital myopathy without available curative treatment and with pathomechanisms still largely unknown. Autosomal-dominant centronuclear myopathy exhibits a wide clinical spectrum from severe-neonatal to mild-adult forms. The most frequent phenotype corresponds to late-childhood or adult onset forms in which motor milestones are delayed and diffuse skeletal muscle weakness mainly involves facial and limb muscles (Fischer et al, 2006; Hanisch et al, 2011). In the severe and early-onset CNM (Bitoun et al, 2007), paediatric patients usually have generalized weakness, hypotonia, facial weakness with open mouth, ptosis and ophthalmoplegia. Few children can improve their strength during early childhood (Susman et al, 2010), but some of them can still develop a restrictive respiratory syndrome at later ages (Bitoun et al, 2007; Melberg et al, 2010). The histological features in muscle biopsies consist of nuclear centralization associated with atrophy and predominance of type 1 fibres and radial arrangement of sarcoplasmic strands radiating from the central nuclei

Methods

Results

Conclusion