CRISPR-inhibition for FSHD

摘要 面肩肱骨肌营养不良症(FSHD)是一种复杂的遗传和表观遗传学 染色体D4Z4大卫星重复序列染色质松弛引起的疾病 4q35，导致DUX4基因在骨骼肌中异常表达和致病。 FSHD治疗的最直接途径是消除DUX4mRNA的表达。 重要的是，来自临床受影响和无症状的FSHD受试者的数据支持任何 减少DUX4的表达将有治疗上的好处。 CRISPR/Cas9技术已被广泛用于修饰特定基因组 这为许多疾病提供了永久纠正的潜力。在危险的同时 与标准CRISPR编辑相关联的是在重复区域中特别令人关注的，例如 在FSHD基因座上，使用CRISPR抑制基因表达是FSHD的理想选择。我们 已经表明，CRISPR抑制(CRISPRi)由死亡的Cas9融合成一个小的 转录抑制物可抑制FSHD心肌细胞中DUX4的表达，为DUX4在FSHD中的表达提供了证据 能够成功地靶向和抑制致病重复的原则。然而，一个 有效的治疗需要有效地将治疗成分输送到骨骼 肌肉和长期压抑的疾病部位。为了满足这些需求，我们重新- 我们设计的CRISPRi平台允许在体内传递更大、更强大的 表观遗传抑制因子。我们设计了一种FSHD优化的调节盒来驱动 金黄色葡萄球菌较小的dCas9同源基因(DSaCas9)与四个不同的表观遗传抑制因子融合 (Hp1、Hp1、MeCP2转录抑制域或SUV39H1SET结构域) 以前太大了，不适合AAV载体。将这些因子靶向DUX4启动子 或者外显子1使FSHD基因座的染色质返回到更正常的抑制状态， 降低DUX4及其靶基因的表达，对肌肉无不良影响 转录组。在这项提案中，我们将对CRISPR进行首次活体评估- 基于治疗FSHD的方法，使用其中所有CRISPRi分量都是 装在单一的治疗盒中。这些将在培养的原代FSHD中进行测试 在两个FSHD小鼠模型中，提供了互补的优势 临床评估。这一提议的前提是AAV介导的dCas9靶向 DUX4的表观遗传抑制子可以有效稳定地沉默疾病基因座 有害的偏离目标的效果。该项目的成功完成将为临床前提供 验证了一种纠正FSHD根本表观遗传失调的治疗方法。

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.