CRISPR-inhibition for FSHD

总结 面肩肱型肌营养不良症（FSHD）是一种复杂的遗传和表观遗传 由染色体上D4 Z4大卫星重复序列的染色质松弛引起的疾病 4 q35，导致骨骼肌中DUX 4基因的异常和致病性表达。 FSHD治疗的最直接途径是消除DUX 4 mRNA的表达。 重要的是，来自临床受累和无症状FSHD受试者的数据支持任何 DUX 4表达的降低将具有治疗益处。 CRISPR/Cas9技术已被广泛用于修饰特定的基因组， 这是一个巨大的挑战，为许多疾病的永久纠正提供了可能。虽然危险 与标准CRISPR编辑相关的基因在重复区域中特别值得关注， 在FSHD基因座中，使用CRISPR来抑制基因表达理想地适合于FSHD。我们 已经表明，由死亡Cas9融合到一个小的CRISPR抑制（CRISPRi）， 转录抑制剂可以抑制FSHD心肌细胞中DUX 4的表达，提供了证据， 致病性重复可以被成功地靶向和抑制的原则。但安 有效治疗需要有效地将治疗组分递送到骨骼 肌肉和疾病位点的长期抑制。为了满足这些需求，我们将- 设计了我们的CRISPRi平台，允许在体内递送更大，更强大的 表观遗传阻遏物我们设计了一种FSHD优化的调控盒， 来自S.金黄色葡萄球菌（dSaCas 9）融合到四种不同的表观遗传阻遏物 (HP1在一些实施方案中，该结构域包括MeCP 2转录抑制结构域（例如，MCP 2、HP 1 β、MeCP 2转录抑制结构域或SUV 39 H1 SET结构域），其 以前太大而不能装入AAV载体。将这些因子靶向DUX 4启动子 或者外显子1使FSHD基因座上的染色质恢复到更正常的抑制状态， 减少DUX 4及其靶基因的表达，而对肌肉没有有害影响 转录组在这项提案中，我们将对CRISPR进行首次体内评估， 使用新载体治疗FSHD的方法，其中所有CRISPRi组分都是 包含在单个治疗盒中。这些将在培养的原代FSHD中进行检测 肌细胞和两个FSHD小鼠模型，提供了互补的优势， 临床评估。该提议的前提是AAV介导的dCas 9靶向 DUX 4的表观遗传阻遏物可以有效和稳定地沉默疾病位点， 有害的脱靶效应该项目的成功完成将提供临床前 验证了纠正FSHD中基本表观遗传失调的治疗。

SUMMARY Facioscapulohumeral muscular dystrophy (FSHD) is a complex genetic and epigenetic disease caused by chromatin relaxation of the D4Z4 macrosatellite repeat array at chromosome 4q35, which leads to aberrant and pathogenic expression of the DUX4 gene in skeletal muscle. The most direct path to an FSHD therapy is eliminating expression of DUX4 mRNA. Importantly, data from clinically affected and asymptomatic FSHD subjects support that any reduction in DUX4 expression will have therapeutic benefit. CRISPR/Cas9 technology has been used extensively to modify specific genomic regions, offering the potential for permanent correction of many diseases. While the dangers associated with standard CRISPR editing are of particular concern in a repetitive region such as the FSHD locus, the use of CRISPR to repress gene expression is ideally suited to FSHD. We have shown that CRISPR inhibition (CRISPRi) consisting of dead Cas9 fused to a small transcriptional inhibitor can repress expression of DUX4 in FSHD myocytes, providing proof-of- principle that the pathogenic repeat can be successfully targeted and repressed. However, an effective therapy will require both efficient delivery of therapeutic components to skeletal muscles and long-term repression of the disease locus. To address these needs, we re- engineered our CRISPRi platform to allow in vivo delivery of larger and more powerful epigenetic repressors. We designed an FSHD-optimized regulatory cassette to drive the smaller dCas9 ortholog from S. aureus (dSaCas9) fused to four different epigenetic repressors (HP1, HP1, the MeCP2 transcriptional repression domain, or the SUV39H1 SET domain) that were previously too large to fit into AAV vectors. Targeting these factors to the DUX4 promoter or exon 1 returns the chromatin at the FSHD locus to a more normal state of repression, reducing expression of DUX4 and its target genes with no deleterious effects on the muscle transcriptome. In this proposal, we will undertake the first in vivo assessment of a CRISPR- based approach to treating FSHD, using new vectors in which all CRISPRi components are contained within single therapeutic cassettes. These will be tested in cultured primary FSHD myocytes and in two FSHD mouse models that provide complementary advantages for pre- clinical assessment. The premise of this proposal is that AAV-mediated dCas9 targeting of epigenetic repressors to DUX4 can effectively and stably silence the disease locus without deleterious off-target effects. Successful completion of this project will provide the preclinical validation for a treatment that corrects the fundamental epigenetic dysregulation in FSHD.