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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

弗里德赖希共济失调 (FRDA) 是最常见的常染色体隐性神经肌肉疾病。由 frataxin (FXN) 基因第一个内含子中的 GAA 重复扩增引起，目前尚无有效治疗方法。由于患者基因组中保留了扩展的重复序列，因此可以进行治疗。我们发现抑制H3K9三甲基化是迫切需要的。 (H3K9me3) 与 DNA 碱基切除修复 (BER) 协同作用以收缩扩展的 GAA 重复序列并上调 FRDA 神经细胞和转基因小鼠大脑中的 FXN 基因表达 FXN 基因处 H3K9me2/me3 的重复靶向去甲基化可破坏异染色质并诱导 BER 为了验证这一假设，我们建议使用 CRISPR/Cas9 系统。组蛋白 H3-三甲基-L-赖氨酸 9 去甲基酶 4D (KDM4D) 与催化灭活的化脓性链球菌 Cas9 融合 (CRISPR/dCas9-KDM4D) 在 FRDA 神经细胞中诱导 H3K9me2/me3 的 GAA 重复靶向去甲基化。我们将追求两个具体目标 1 是确定 GAA 重复靶向 CRISPR/dCas9-KDM4D 是否可以。去甲基化 H3K9me2/me3 以破坏 FRDA 神经细胞中 FXN 基因的异染色质。使用质粒 pCRISPR/dCas9-DNMT3A-PuroR_v2 将人类 KDM4D 基因与化脓性链球菌 dCas9 结合作为主链，KDM4D 将通过 XTEN80 连接链连接到 dCas9 的 C 末端。用于编码单链引导RNA (sgRNA) 的序列，该单链引导RNA 靶向5'-或3'-侧翼区域扩增的 GAA 重复序列也将被插入到质粒中。质粒将被稳定转染到 FRDA 中。神经祖细胞 (NPC) 从 FRDA 患者的诱导多能干细胞 (iPSC) 分化而来。其次，我们将确定重复靶向的 dCas9-KDM4D 是否可以降低 H3K9me2/me3 的水平和从 NPC 分化而来的 FRDA 神经细胞中扩增重复序列的异染色质化。目的是确定针对 GAA 重复的 CRISPR/dCas9-KDM4D 是否通过以下方式促进 GAA 重复收缩： BER，导致 FXN 基因表达上调并减轻线粒体功能障碍首先，我们将确定 dCas9-KDM4D 是否可以导致 GAA 重复收缩。确定 dCas9-KDM4D 是否可以促进关键 BER 酶 DNA 聚合酶 β (Pol β) 的招募，和 FLAG 核酸内切酶 1 (FEN1) 到 FRDA 神经细胞中的扩展重复序列其次，我们将测试 dCas9- 是否有效。 KDM4D 可导致 FXN 基因表达上调并缓解线粒体功能障碍。研究将为通过协同作用对扩展的 GAA 重复序列进行基因靶向收缩提供概念证明研究结果将揭示组蛋白修饰和 DNA 修复之间的潜在机制。 CRISPR/dCas9-KDM4D 通过组蛋白的相互作用靶向扩展 GAA 重复序列的收缩该研究将进一步为开发有效的 FRDA 基因治疗开辟新途径。