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 面肩肱型肌营养不良症（FSHD）是一种复杂的遗传和表观遗传性疾病 由染色体 D4Z4 大卫星重复序列染色质松弛引起的疾病 4q35，导致骨骼肌中 DUX4 基因的异常和致病性表达。 FSHD 治疗最直接的途径是消除 DUX4 mRNA 的表达。 重要的是，来自临床受影响和无症状 FSHD 受试者的数据支持任何 DUX4 表达的减少将具有治疗益处。 CRISPR/Cas9技术主要用于修饰特定基因组 地区，提供了永久纠正许多疾病的潜力。 与标准 CRISPR 编辑相关的重复区域尤其值得关注，例如 在 FSHD 基因座上，使用 CRISPR 抑制基因表达非常适合 FSHD。 已表明 CRISPR 抑制 (CRISPRi) 由死亡的 Cas9 与小片段融合组成 转录抑制剂可以抑制 FSHD 肌细胞中 DUX4 的表达，提供了证据- 可以成功地靶向和抑制致病重复原理。 有效的治疗需要将治疗成分有效递送至骨骼 肌肉和长期压抑的疾病部位。为了解决这些需求，我们重新 设计了我们的 CRISPRi 平台，可以在体内递送更大、更强大的 我们设计了一个 FSHD 优化的调控盒来驱动 来自金黄色葡萄球菌 (dSaCas9) 的较小 dCas9 直系同源物与四种不同的表观遗传阻遏蛋白融合 （HP1、HP1、MeCP2 转录抑制结构域或 SUV39H1 SET 结构域） 之前太大，无法放入 AAV 载体中，将这些因子靶向 DUX4 启动子。 或外显子 1 将 FSHD 位点处的染色质恢复到更正常的抑制状态， 减少 DUX4 及其靶基因的表达而不对肌肉产生有害影响 在本提案中，我们将对 CRISPR 进行首次体内评估。 基于治疗 FSHD 的方法，使用新载体，其中所有 CRISPRi 组件都在 这些将在培养的原代 FSHD 中进行测试。 肌细胞和两个 FSHD 小鼠模型，为预-提供互补的优势 该提案的前提是 AAV 介导的 dCas9 靶向。 DUX4的表观遗传阻遏物可以有效、稳定地沉默疾病位点，而无需 该项目的成功完成将提供临床前的有害影响。 验证纠正 FSHD 基本表观遗传失调的治疗方法。