Facioscapulohumeral muscular dystrophy (FSHD) is caused by incomplete silencing of the disease locus, leading to pathogenic misexpression of DUX4 in skeletal muscle. Previously, we showed that CRISPR inhibition could successfully target and repress DUX4 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 in vivo delivery of more potent epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive skeletal muscle-specific expression of dCas9 from Staphylococcus aureus fused to HP1α, HP1γ, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain. Targeting each regulator to the DUX4 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 in vivo 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.

面肩肱型肌营养不良症 (FSHD) 是由疾病基因座的不完全沉默引起的，导致骨骼肌中DUX4 的致病性错误表达。以前，我们表明 CRISPR 抑制可以成功地靶向和抑制FSHD 肌细胞中的DUX4。然而，有效的治疗既需要有效地将治疗成分输送到骨骼肌，也需要长期抑制疾病部位。因此，我们重新设计了我们的平台，以允许在体内递送更有效的表观遗传阻遏物。我们设计了一个 FSHD 优化的调节盒来驱动来自金黄色葡萄球菌的 dCas9 的骨骼肌特异性表达与 HP1α、HP1γ、MeCP2 转录抑制域或 SUV39H1 SET 域融合。将每个调节因子靶向DUX4启动子/外显子 1 会增加该基因座的染色质抑制，特别是抑制 FSHD 肌细胞和疾病小鼠模型中的 DUX4 及其靶基因。重要的是，最小化调节盒并使用较小的 Cas9 直向同源物使我们的治疗盒能够有效地包装到腺相关病毒 (AAV) 载体中以进行体内递送。通过设计与 AAV 载体兼容的肌肉特异性表观遗传 CRISPR 平台用于基因治疗，我们为基于 dCas9 的染色质效应器在骨骼肌疾病中的临床应用奠定了基础。