Targeted epigenetic repression by CRISPR/dSaCas9 suppresses pathogenic expression in FSHD.

Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress in FSHD myocytes. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. Thus, we re-engineered our platform to allow delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the promoter/exon 1 increased chromatin repression at the locus, specifically suppressing DUX4 and its target genes in FSHD myocytes and in a mouse model of the disease. Importantly, minimizing the regulatory cassette and using the smaller Cas9 ortholog allowed our therapeutic cassettes to be effectively packaged into adeno-associated virus (AAV) vectors for delivery. By engineering a muscle-specific epigenetic CRISPR platform compatible with AAV vectors for gene therapy, we have laid the groundwork for clinical use of dCas9-based chromatin effectors in skeletal muscle disorders.