Table 1 |.
Targeted therapies for facioscapulohumeral muscular dystrophy
| Agent | Mechanism | Preclinical and clinical findings | Refs. |
|---|---|---|---|
| Small molecules | |||
| β2 Adrenergic receptor agonists | Presumed to inhibit DUX4 transcription by promoting muscle hypertrophy, in part through modulating cAMP levels | ↓DUX4 mRNA, ↓DUX4 target gene expression and ↑intracellular cAMP in FSHD patient-derived muscle cells RCTs in 90 and 65 patients with FSHD: ↑lean muscle mass, ↑grip strength; no change or limited improvement in maximum voluntary isometric strength in some muscles |
166–168 |
| BET protein BRD4 inhibitor | Inhibition of DUX4 transcription blocks BRD4- dependent recruitment of transcription regulators to D4Z4 | ↓DUX4 mRNA and ↓ DUX4 target gene expression in FSHD patient-derived muscle cells | 168 |
| p38 MAPK inhibitors (losmapimod) | Inhibition of p38 MAPK signalling has anti-inflammatory effects. Decreased DUX4 expression observed through unknown mechanism | ↓DUX4 mRNA and ↓DUX4 target gene expression in FSHD patient-derived muscle cells; ↓DUX4 and DUX4 target gene expression in a mouse FSHD xenograft model Phase 2b RCT in 80 patients with FSHD (awaits peer review): improvement in some patient-reported outcomes albeit without DUX4 attenuation (possibly through anti-inflammatory properties); phase 3 trial planned |
169–173 |
| P300 histone acetyltransferase inhibitor (iP300w) | Inhibits DUX4-mediated recruitment of p300–CBP to DUX4 target genes by blocking p300 | ↓DUX4 target gene expression; ↓global acetylated histone H3 abundance (accessible chromatin marker) in FSHD myotubes and in a transgenic FSHD mouse model | 190 |
| Oligotherapeutics | |||
| PMO and/or AON targeting 3’-UTR of DUX4 pre-mRNA (PAS and/or cleavage sites) | Prevents correct pre-mRNA processing (intron splicing or polyadenylation), inhibiting mRNA stability and promoting transcript degradation | ↓DUX4 mRNA and ↓DUX4 target gene expression; cleavage site redirection that rescued DUX4 observed with one PMO; ↓DUX4 protein and ↓atrophic myotubes; ↑muscle mass and strength; ↓myofibre central nucleation and muscle fibrosis; in FSHD patient-derived muscle cells, FSHD patient-derived primary muscle cells and in FSHD xenograft and/or transgenic FSHD mouse models | 179–184 |
| AON targeting exon 1 of DUX4 transcript | Inhibits DUX4 mRNA translation, promoting transcript degradation | ↓DUX4 mRNA, DUX4 protein and DUX4 target gene expression; ↓skeletal muscle pathology; improved treadmill test results; no improvement in strength test results in a transgenic FSHD mouse model | 185 |
| siRNAs bearing sequence homology to DUX4 | Exogenous siRNA targeting the 5’-UTR of DUX4 pre-mRNA | ↓DUX4 mRNA; ↑H3K9me2 and recruitment of AGO2 (indicating RNA-mediated epigenetic silencing) in FSHD patient-derived muscle cells | 176 |
| miR-405 | RNA interference through artificial miRNA targeting human DUX4 mRNA open reading frame, inhibiting transcription | ↓DUX4 mRNA in luciferase assay screen; ↓skeletal muscle pathology but no notable improvement in grip strength with AAV-miDUX4.405 overexpression in the AAV-DUX4-transgenic mouse model | 187,188 |
| miR-675 | RNA interference through naturally occurring human miR-675 directly targeting both the 3’-UTR and open reading frame of DUX4 mRNA | ↓DUX4 mRNA, ↓DUX4 protein, ↓DUX4 target gene expression, ↓DUX4-induced cell death; ↓transactivation of DUX4-responsive reporter in HEK293T cells transfected with DUX4 and in FSHD patient-derived muscle cells; ↓skeletal muscle pathology in transgenic (AAV-DUX4 induced) FSHD mouse model | 177 |
| U7 small nuclear (sn) RNA antisense expression cassettes | Alters target specificity of sn ribonucleoprotein complex to target DUX4 pre-mRNA, inhibiting its maturation | ↓DUX4-induced cell death; ↓DUX4 mRNA; ↓DUX4 protein in transfected HEK293 cells overexpressing DUX4; ↓DUX4 mRNA and ↓DUX4 target gene expression in FSHD patient-derived muscle cells | 189 |
| Double-stranded (ds) DNA oligonucleotides encoding DUX4 binding sites | Inhibits DUX4 transactivational activity through competition of decoy binding sites with real DUX4 target gene-binding sites | ↓DUX4 target gene expression on direct electrotransfection of dsDNA in FSHD patient-derived muscle cells and on systemic delivery of AAV-dsDNA decoy in mice overexpressing DUX4 from transfected plasmid vector | 191 |
| DNA aptamers | Bind DUX4 protein with increased affinity and specificity, inhibiting DUX4 target binding and activation | Aptamers enhance affinity and specificity of decoy binding site sequences for DUX4 using recombinant DUX4 (SELEX-derived aptamers have not yet been tested in DUX4-expressing cells) | 192 |
| Gene editing | |||
| CRISPR–Cas9 | Restores SMCHD1 reading frame by removing an intronic mutation causing a cryptic splice site | ↑Wild-type SMCHD1; ↓DUX4 mRNA; ↓DUX4 target gene expression in FSHD2 patient-derived muscle cells; no effect on D4Z4 DNA methylation | 77 |
| CRISPR–Cas9 | CRISPR–Cas9 and TALEN-based elimination of DUX4 PAS (exon 3) | ↓DUX4 mRNA and ↓DUX4 target gene expression in double-knockout HCT116 (human colorectal cancer cell) model; use of alternative PAS upstream of targeted PAS in FSHD patient-derived muscle cells | 198 |
| CRISPR–Cas9 | CRISPR–Cas9 elimination of DUX4 PAS (exon 3) | ↓DUX4 mRNA and ↓DUX4 target gene expression in double-knockout HCT116 human colorectal cancer cell model and in FSHD patient-derived muscle cells | 199 |
| CRISPR–dCas9 adenine base editor | Induces mutation (AT → CG conversion) in DUX4 PAS | ↓DUX4 mRNA and ↓DUX4 target gene expression in FSHD patient-derived muscle cells | 203 |
| CRISPR–dCas9 inhibition | |||
| CRISPR–dCas9 transcriptional repression | CRISPR–dCas9 orthologue fused to transcriptional repressors of DUX4 | ↓DUX4 mRNA and ↓DUX4 target gene expression in FSHD primary myocytes; ↓DUX4 and DUX4 target gene expression in an FSHD transgenic mouse model (more modest repression) | 204 |
| CRISPR–dCas9–KRAB transcriptional repression | KRAB-repressor targeting the DUX4 gene promoter | ↓DUX4 mRNA and ↓DUX4 target gene expression in FSHD primary myocytes; no effect on expression of other genes in the D4Z4 locus (FRG1 and FRG2) | 197 |
| CRISPR–dCas9–KRAB transcriptional repression | KRAB-repressor targeting DUX4 transcription activators | ↓DUX4 mRNA; ↑H3K9me3 (evidence of altered chromatin) at the targeted D4Z4 locus in FSHD primary myocytes | 147 |
| CRISPR–dCas9–KRAB transcriptional repression | KRAB-repressor targeting the DUX4 PAS | ↓DUX4 mRNA and DUX4 target gene expression; (partial) restoration of repressive H3K9me3 in FSHD patient-derived muscle cells | 199 |
AAV, adeno-associated virus; AGO2, argonaute RISC catalytic component 2; AON, antisense oligonucleotide; BET, bromodomain and extra terminal domain; BRD4, bromodomain-containing protein 4; cAMP, cyclic adenosine monophosphate; Cas9, caspase 9; CBP, cyclic adenosine monophosphate response element binding protein (CREB) binding protein; CRISPR, clustered regularly interspaced short palindromic repeat; dCas9, ‘dead’ (DNA cleavage-inactivated) Cas9; DUX4, double homeobox protein 4; FSHD, facioscapulohumeral muscular dystrophy; KRAB, Krüppel-associated box; MAPK, mitogen-activated protein kinase; PAS, polyadenylation signal; PMO, phosphorodiamidate morpholino oligomer; RCT, randomized controlled trial; SELEX, systematic evolution of ligands by exponential enrichment; siRNA, small interfering RNA; TALEN, transcription activator-like effector nucleases; UTR, untranslated region of pre-mRNA; mRNA, messenger RNA; miR, microRNA; SMCHD1, structural maintenance of chromosomes flexible hinge domain containing protein 1.