CRISPR-inhibition for FSHD

FSHD 的 CRISPR 抑制

• 批准号: 10318054
• 负责人: Peter L Jones
• 金额: $ 53.89万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-04 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10318054
• 关键词: 4q35; Address; Affect; Alleles; Animal Model; CRISPR/Cas technology; Cells; Chromatin; Chromosomes; Clinical Data; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; D4Z4; Disease; Distal; Engineering; Epigenetic Process; Exons; Facioscapulohumeral Muscular Dystrophy; Gene Expression; Genes; Genetic; Genetic Transcription; Genome; Genomic Segment; Human; Mammals; Mediating; Messenger RNA; Methyl-CpG-Binding Protein 2; Modeling; Mus; Muscle; Muscle Cells; Myopathy; Orthologous Gene; Pathogenesis; Pathogenicity; Pathology; Pathway interactions; Patients; Phenotype; Primates; Relaxation; Repetitive Sequence; Repression; SET Domain; Skeletal Muscle; Specificity; Staphylococcus aureus; Technology; Testing; Therapeutic; Transgenic Mice; Validation; Viral Load result; Xenograft Model; Xenograft procedure; adeno-associated viral vector; base; design; effective therapy; efficacy testing; efficacy validation; gene repression; improved; in vivo; in vivo evaluation; inhibitor/antagonist; mouse model; muscular dystrophy mouse model; novel; pre-clinical; pre-clinical assessment; promoter; transcriptome; vector

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.

概括 Facioscapulohumeral肌肉营养不良（FSHD）是一种复杂的遗传和表观遗传学 D4Z4宏观卫星重复阵列的染色质松弛引起的疾病 4Q35，导致骨骼肌中Dux4基因的异常和致病表达。 FSHD治疗的最直接途径是消除DUX4 mRNA的表达。 重要的是，来自临床受影响和不对称FSHD受试者的数据支持任何 DUX4表达的降低将具有治疗益处。 CRISPR/CAS9技术已广泛用于修改特定的基因组 地区，提供了许多疾病的永久性纠正的潜力。而危险 在重复区域（例如 FSHD基因座，使用CRISPR抑制基因表达非常适合FSHD。我们 已经表明，CRISPR抑制（CRISPRI）由死亡Cas9组成 转录抑制剂可以反映DUX4在FSHD肌细胞中的表达，从而提供证明 可以成功靶向和再现病原重复的原理。但是，一个 有效的治疗将需要有效地递送治疗成分到骨骼 肌肉和疾病基因座的长期反射。为了满足这些需求，我们重新 设计了我们的CRISPRI平台，以允许体内交付更大，更强大的 表观遗传复制品。我们设计了一个FSHD优化的调节盒，以驱动 较小的DCAS9直系同源物来自金黄色葡萄球菌（DSACAS9），融合到四个不同的表观遗传复制品 （HP1，HP1，MECP2转录表达域或SUV39H1设置域） 以前太大了，无法适应AAV矢量。将这些因素靶向DUX4启动子 或外显子1将FSHD基因座的染色质返回到更正常的表达状态， 减少DUX4及其靶基因的表达，对肌肉没有有害影响 转录组。在这项提案中，我们将对CRISPR-的第一个体内评估进行 基于处理FSHD的方法，使用所有CRISPRI组件的新向量 包含在单个治疗盒中。这些将在培养的主要FSHD中进行测试 心肌细胞和两种FSHD小鼠模型，它们为预审长提供了完整的优势 临床评估。该提议的前提是AAV介导的DCAS9针对 对DUX4的表观遗传学复制品可以有效而稳定地使疾病基因座保持不存在 有害的脱靶效果。该项目的成功完成将提供临床前 验证纠正FSHD中基本表观遗传失调的治疗方法。