MicroRNA-based epigenetic approach to induce fetal hemoglobin

基于 MicroRNA 的表观遗传学方法诱导胎儿血红蛋白

• 批准号: 10212449
• 负责人: Athena Starlard-Davenport
• 金额: $ 30.53万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-13 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10212449
• 关键词: 3' Untranslated Regions; ANXA5 gene; Address; Adult; Biological Assay; Blood Cell Count; CD34 gene; Cell Proliferation; Cells; Child; Cholesterol; Clinical; Complete Blood Count; Continuous Infusion; Cytosine; DNA; DNA Damage; DNA Methylation; DNA Methyltransferase Inhibitor; DNA Modification Methylases; DNA Sequence Alteration; Data; Development; Differentiation Antigens; Disease; Dose; Enzymes; Epigenetic Process; Erythroid; Erythropoiesis; FDA approved; Fertility; Fetal Hemoglobin; Flow Cytometry; Fluorescence; Functional disorder; GYPA gene; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Gene Silencing; Genetic Transcription; Genome; Genotype; Globin; Goals; Hematopoiesis; Hemoglobinopathies; Human; Hypermethylation; Hypoxia; In Vitro; Individual; Infant; Infusion procedures; Knowledge; Liquid substance; MYB gene; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Methylation; MicroRNAs; Molecular; Monitor; Mus; Mutation; Oncogenes; Patients; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiological; Plasmids; Play; Population; Propidium Diiodide; Publishing; Pump; Regulation; Research; Reticulocyte count; Role; Severity of illness; Sickle Cell; Sickle Cell Anemia; Spleen; Subcutaneous Injections; Switch Genes; Symptoms; System; TFRC gene; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfection; Transferase; Treatment Efficacy; Untranslated RNA; Work; base; bench to bedside; beta Globin; cancer cell; chromatin modification; drug development; efficacy testing; epigenetic silencing; erythroid differentiation; gamma Globin; hydroxyurea; in vivo; innovation; methylation pattern; mouse model; novel; novel therapeutics; overexpression; pre-clinical; prevent; progenitor; promoter; sickling; side effect; small molecule; stem cells; subcutaneous; transcriptome sequencing

Project Abstract This SHINE II application seeks to explore epigenetic mechanisms that control globin gene regulation during erythropoiesis. The central hypothesis of this project is that that miR-29b reverses the γ-globin to β-globin gene switch and induces HbF via modulation of DNA methylation. Therapeutic interventions aimed at inducing HbF expression is an effective approach for ameliorating the clinical symptoms of sickle cell disease (SCD) in adults and children. Hydroxyurea is the only FDA-approved drug with proven efficacy for inducing HbF in patients with SCD, but DNA methyltransferase (DNMT) inhibitors have shown promise as HbF inducers by producing proximal γ-globin promoter DNA hypomethylation. However, DNMT inhibitors can produce off-target side effects. Small non-coding microRNAs (miR) are attractive molecules for targeting repressors of γ-globin gene expression and studies show that miR-29b inhibits DNA methylation through direct targeting of the 3’ untranslated region of DNMT3A and DNMT3B. The objective of this proposal is to test the efficacy of miR-29b as an HbF inducer. Our published work shows that miR-29b is important for reactivating γ-globin transcription and HbF expression by targeting MYB, a known repressor of γ-globin involved in mediating DNA methylation and gene silencing. To test our central hypothesis, we will accomplish one specific aim to determine the ability of miR-29b to mediate epigenetic changes in DNA methylation in the HBB locus and reactivate γ-globin transcription and HbF expression during adult erythropoiesis. Sub-aim A will explore the molecular effects of miR-29b on γ-globin regulation using an in vitro primary liquid culture system to generate erythroid progenitors from peripheral blood mononuclear cells isolated from individuals with SCD. These findings will compare to normal erythroid progenitors to validate the ability of miR-29b to reactivate γ-globin transcription through proximal promoter DNA methylation. Off-target effects mediated by miR-29b will be investigated by DNA mutation and RNA-seq analyses. To define the role of miR-29b in reversing phenotype, erythroid progenitor sickling under hypoxic conditions will be evaluated. Sub-aim B will establish the optimal dose of miR-29b that mediates γ-globin reactivation and HbF induction in vivo using a preclinical Townes SCD mouse model. This research highlights a novel miRNA-based epigenetic approach to induce HbF to impact the discovery of new drugs to expand treatment options for SCD.

项目摘要 这个SHINE II应用程序旨在探索控制珠蛋白基因的表观遗传机制 在红细胞生成过程中的调节。该项目的中心假设是， 逆转γ-珠蛋白到β-珠蛋白的基因开关，并通过DNA调节诱导HbF 甲基化旨在诱导HbF表达的治疗干预是一种有效的方法 用于改善成人和儿童的镰状细胞病（SCD）的临床症状。 羟基脲是FDA批准的唯一一种经证实有效的药物，可用于诱导HbF， SCD，但DNA甲基转移酶（DNMT）抑制剂已显示出作为HbF诱导剂的前景， 产生近端γ-珠蛋白启动子DNA低甲基化。然而，DNMT抑制剂可以 产生脱靶副作用小的非编码microRNA（miR）是对细胞生长有吸引力的分子。 靶向γ-珠蛋白基因表达的阻遏物，研究表明miR-29 b抑制DNA 通过直接靶向DNMT 3A和DNMT 3B的3'非翻译区，可以使DNA甲基化。的 该提议的目的是测试miR-29 b作为HbF诱导剂的功效。我们已发表的作品 显示miR-29 b对于通过以下途径重新激活γ-珠蛋白转录和HbF表达是重要的： 靶向MYB，一种已知的参与介导DNA甲基化和基因转移的γ-珠蛋白阻遏物， 沉默为了检验我们的中心假设，我们将完成一个特定的目标，以确定 miR-29 b介导HBB基因座中DNA甲基化的表观遗传变化的能力， 在成人红细胞生成过程中重新激活γ-珠蛋白转录和HbF表达。次级目标A将 使用体外原液探索miR-29 b对γ-珠蛋白调节的分子效应 从分离的外周血单核细胞产生红系祖细胞的培养系统 患有SCD的人。这些发现将与正常红系祖细胞进行比较，以验证 miR-29 b通过近端启动子DNA重新激活γ-珠蛋白转录能力 甲基化miR-29 b介导的脱靶效应将通过DNA突变和 RNA-seq分析为了确定miR-29 b在逆转表型中的作用， 将评估缺氧条件下的镰状化。子目标B将确定 使用临床前Townes方法在体内介导γ-珠蛋白再活化和HbF诱导的miR-29 b SCD小鼠模型。这项研究强调了一种新的基于miRNA的表观遗传方法， HbF影响新药的发现，以扩大SCD的治疗选择。