A CRSIPR/dCas9-Targeted Histone Demethylation Induces GAA repeat contraction

CRSIPR/dCas9 靶向组蛋白去甲基化诱导 GAA 重复收缩

• 批准号: 10649032
• 负责人: Yuan Liu
• 金额: $ 7.38万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10649032
• 关键词: 3' Flanking Region; 8-hydroxyguanosine; Aconitate Hydratase; Advanced Development; Alkylating Agents; Ataxia; Base Excision Repairs; Biological Assay; Brain; Cardiomyopathies; Cell Differentiation Induction; Cell Differentiation process; Cells; Chromatin; Clone Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Contracts; DNA; DNA Double Strand Break; DNA Modification Process; DNA Polymerase beta; DNA Repair; DNA Sequence; DNMT3a; Disease; Epigenetic Process; Excision Repair; Friedreich Ataxia; Gene Expression; Gene Silencing; Gene Targeting; Genes; Genome; Genome Stability; Genomic Instability; Guide RNA; Heterochromatin; Histone H3; Histones; Human; Introns; Iron; Link; Lobe; Lysine; Maps; Mediating; Messenger RNA; Methylation; Mission; Neurodegenerative Disorders; Neuromuscular Diseases; Neurons; Oxidative Stress; Pathway interactions; Patients; Phenotype; Plasmids; Production; Proteins; RTH-1 Nuclease; Research; Streptococcus pyogenes; Sulfur; System; Testing; Tissues; Transcription Elongation; Transfection; Transgenic Mice; United States National Institutes of Health; Up-Regulation; Vertebral column; autosome; demethylation; disability; effective therapy; frataxin; gene therapy; histone demethylase; histone methylation; histone modification; induced pluripotent stem cell; insight; mitochondrial dysfunction; nerve stem cell; neural; novel; novel strategies; public health relevance; recruit; repair enzyme; synergism; temozolomide

Friedreich’s Ataxia (FRDA) is the most common autosomal recessive neuromuscular disorder. The disease is caused by expanded GAA repeats in the first intron of the frataxin (FXN) gene. No effective treatments for the disease are available, owing to the expanded repeats remaining in the patients’ genome. Thus, a treatment that targets the expanded GAA repeats is urgently needed. We found that the inhibition of H3K9 trimethylation (H3K9me3) synergized with DNA base excision repair (BER) to contract the expanded GAA repeats and upregulate FXN gene expression in FRDA neural cells and transgenic mouse brain. We hypothesize that GAA repeat-targeted demethylation of H3K9me2/me3 at the FXN gene can disrupt heterochromatin and induce BER to contract the expanded repeats. To test this hypothesis, we propose to use a CRISPR/Cas9 system with the histone H3-trimethyl-L-Lysine 9 demethylase 4D (KDM4D) fused to catalytically inactivated S. pyogenes Cas9 (CRISPR/dCas9-KDM4D) to induce GAA repeat-targeted demethylation of H3K9me2/me3 in FRDA neural cells. We will pursue two Specific Aims. Aim 1 is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D can demethylate H3K9me2/me3 to disrupt heterochromatin at the FXN gene in FRDA neural cells. First, we will fuse the human KDM4D gene with the S. pyogenes dCas9 using the plasmid pCRISPR/dCas9-DNMT3A-PuroR_v2 as a backbone. KDM4D will be linked to the C-terminus of dCas9 through the XTEN80 linker chain. The sequences for coding the single-strand guide RNAs (sgRNAs) that target the 5’- or 3’-flanking regions of the expanded GAA repeats will also be inserted into the plasmid. The plasmid will be stably transfected into FRDA neural progenitor cells (NPCs) differentiated from induced pluripotent stem cells (iPSCs) of an FRDA patient. Second, we will determine if the repeat-targeted dCas9-KDM4D can reduce the level of H3K9me2/me3 and alleviate heterochromatinization on the expanded repeats in FRDA neural cells differentiated from NPCs. Aim 2 is to determine if the GAA repeat-targeted CRISPR/dCas9-KDM4D promotes GAA repeat contraction through BER, leading to the upregulation of the FXN gene expression and the alleviation of mitochondrial dysfunction in FRDA neural cells. First, we will determine if dCas9-KDM4D can lead to GAA repeat contraction. We will then determine if dCas9-KDM4D can facilitate the recruitment of the key BER enzymes, DNA polymerase β (Pol β), and flap endonuclease 1 (FEN1) to the expanded repeats in FRDA neural cells. Second, we will test if dCas9- KDM4D can result in the upregulation of the FXN gene expression and alleviate mitochondrial dysfunction. Our study will provide proof of concept for a gene-targeted contraction of expanded GAA repeats via the synergy between histone modifications and DNA repair. The results will reveal the mechanisms underlying CRISPR/dCas9-KDM4D targeted contractions of expanded GAA repeats through the interplay of histone demethylation with BER. The study will further open a new avenue to develop effective gene therapy for FRDA.

Friedreich的共济失调（FRDA）是最常见的常染色体隐性神经肌肉疾病。疾病是 由Frataxin（FXN）基因的第一内含子中的GAA重复膨胀引起。没有有效的治疗方法 由于患者基因组中剩余的重复量扩大，因此可以使用疾病。那是一种治疗 迫切需要目标扩展的GAA重复序列。我们发现抑制H3K9三甲基化 （H3K9me3）与DNA基本惊喜维修（BER）协同合同，以收缩扩展的GAA重复和 上调FXN基因表达在FRDA神经细胞和转基因小鼠脑中。我们假设GAA 在FXN基因处的H3K9me2/ME3的重复靶向脱甲基化可以破坏异染色质并诱导BER 收缩扩展的重复。为了检验这一假设，我们建议将CRISPR/CAS9系统与 Hisstone H3-三甲基-L-赖氨酸9脱甲基酶4D（KDM4D）融合成催化失活的Pyogenes cas9 （CRISPR/DCAS9-KDM4D）在FRDA神经细胞中诱导H3K9me2/ME3的GAA重复靶向脱甲基化。 我们将追求两个具体的目标。 AIM 1是确定GAA重复定位的CRISPR/DCAS9-KDM4D是否可以 脱甲基H3K9ME2/ME3在FRDA神经细胞中的FXN基因上破坏异染色质。首先，我们将融合 使用质粒PCRISPR/DCAS9-DNMT3A-PUROR_V2使用链球菌DCAS9的人类KDM4D基因 作为骨干。 KDM4D将通过XTEN80接头链链接到DCAS9的C端。这 针对5'-或3'FALKing区域的单链指南RNA（SGRNA）的序列 扩展的GAA重复序列也将插入质粒中。质粒将稳定转换为FRDA 神经祖细胞（NPC）与FRDA患者的诱导多能干细胞（IPSC）区分开。 其次，我们将确定重复定位的DCAS9-KDM4D是否可以降低H3K9me2/ME3的水平 在与NPC区分的FRDA神经细胞中扩展的重复序列上减轻异染色性。目标2 是为了确定GAA重复定位的CRISPR/DCAS9-KDM4D是否会通过 BER，导致FXN基因表达的上调和缓解线粒体功能障碍 FRDA神经细胞。首先，我们将确定DCAS9-KDM4D是否会导致GAA重复收缩。然后我们会 确定DCAS9-KDM4D是否可以促进关键BER酶，DNA聚合酶β（POLβ）， 将核酸内切酶1（FEN1）与FRDA神经细胞的扩展重复序列。其次，我们将测试是否dcas9-- KDM4D可以导致FXN基因表达的上调并减轻线粒体功能障碍。我们的 研究将为通过协同作用的扩展GAA重复的基因靶向收缩提供概念证明 在组蛋白修饰和DNA修复之间。结果将揭示基础机制 CRISPR/DCAS9-KDM4D通过组蛋白的相互作用的扩展GAA重复的目标收缩 脱甲基与Ber化。这项研究将进一步开辟新的途径，以开发有效的FRDA基因疗法。