Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics

Abstract

Derepression of DUX4 in skeletal muscle has emerged as a likely cause of pathology in facioscapulohumeral muscular dystrophy (FSHD). Here we report on the use of antisense phosphorodiamidate morpholino oligonucleotides to suppress DUX4 expression and function in FSHD myotubes and xenografts. The most effective was phosphorodiamidate morpholino oligonucleotide FM10, which targets the polyadenylation signal of DUX4. FM10 had no significant cell toxicity, and RNA-seq analyses of FSHD and control myotubes revealed that FM10 down-regulated many transcriptional targets of DUX4, without overt off-target effects. Electroporation of FM10 into FSHD patient muscle xenografts in mice also down-regulated DUX4 and DUX4 targets. These findings demonstrate the potential of antisense phosphorodiamidate morpholino oligonucleotides as an FSHD therapeutic option.

Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

“…This RNA-seq study indicated that 96% of the genes upregulated in FSHD myotubes were at least partly reduced by the PAS-PMO, while 89% of the mRNAs that were significantly reduced had a least some upregulation in FSHD myotubes. The inhibition was dose-dependent with 50% reduction of footprint gene expression in the 1–3 µmol/L PMO concentration range [74]. The inhibition was also obtained in a model of FSHD muscle xenograft in an immunosuppressed mouse with electroporation-mediated PMO administration [74].…”

“…The inhibition was dose-dependent with 50% reduction of footprint gene expression in the 1–3 µmol/L PMO concentration range [74]. The inhibition was also obtained in a model of FSHD muscle xenograft in an immunosuppressed mouse with electroporation-mediated PMO administration [74]. …”

“…Two research groups independently developed AOs to target DUX4 pre-mRNA elements involved in its cleavage or polyadenylation in order to suppress the protein expression [71,74]. They selected the phosphorodiamidate morpholino (PMO) chemistry for its lack of toxicity in clinical trials for DMD.…”

“…Among the different PMOs, the most efficient targeted the PAS on DUX4 mRNA, and its 25-nt sequence turned out to be identical for both groups, and was also found in our study of a larger AO set (see below). DUX4 suppression was evaluated either by quantitation of several of its footprint gene mRNAs that constitute FSHD biomarkers [71] or by a differential transcriptome study (RNA-seq) of FSHD or healthy myoblasts treated or not with the PMO [74]. This RNA-seq study indicated that 96% of the genes upregulated in FSHD myotubes were at least partly reduced by the PAS-PMO, while 89% of the mRNAs that were significantly reduced had a least some upregulation in FSHD myotubes.…”

Préclinique

“…DUX4 est donc une cible de choix pour les différentes approches thérapeutiques de la DMFSH. Dans cette étude [1], l'analyse RNASeq de cellules de patients et de contrôles familiaux a permis l'identification de cibles transcriptionnelles directes et indirectes de DUX4, servant ultérieurement de biomarqueurs de l'activité de DUX4, car la mesure directe des mRNA de DUX4 et de la protéine est plus déli-cate et aléatoire du fait de la présence de nombreux homologues. Plusieurs oligonucléotides antisens basés sur la chimie des phosphorodiamidates de morpholinos (PMOs) ont été testés pour leur efficacité d'inhibition in vitro, en cultures de myotubes.…”