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 这种Shine II应用程序旨在探索控制球蛋白基因的表观遗传机制 红细胞生成期间的调节。该项目的中心假设是mir-29b 逆转γ-球蛋白为β-珠蛋白基因开关，并通过调节DNA诱导HBF 甲基化。旨在诱导HBF表达的治疗干预措施是一种有效的方法 用于改善成人和儿童镰状细胞疾病（SCD）的临床症状。 羟基脲是唯一一种由FDA批准的药物 SCD，但DNA甲基转移酶（DNMT）抑制剂已显示为HBF诱导剂的希望 产生近端γ-球蛋白启动子DNA低甲基化。但是，DNMT抑制剂可以 产生脱靶副作用。小型非编码microRNA（miR）是有吸引力的分子 靶向γ-珠蛋白基因表达的反射镜和研究表明，miR-29b抑制了DNA 通过直接靶向DNMT3A和DNMT3B的3'未翻译区域的甲基化。这 该建议的目的是测试MiR-29b作为HBF诱导的效率。我们发表的作品 表明miR-29b对于通过重新激活γ-球蛋白转录和HBF表达很重要 靶向MYB，是参与介导DNA甲基化和基因的γ-球蛋白的已知复制品 沉默。为了检验我们的核心假设，我们将实现一个具体目标，以确定 miR-29b介导HBB基因座DNA甲基化表观遗传变化的能力和 在成年红细胞生成期间重新激活γ-球蛋白转录和HBF表达。遗嘱 使用体外原代液体探索miR-29b对γ-珠蛋白调节的分子作用 培养系统以分离的外周血单核细胞产生红细胞祖细胞 来自患有SCD的人。这些发现将与正常的红细胞祖细胞相比 miR-29b通过代理启动子DNA重新激活γ-球蛋白转录的能力 甲基化。由miR-29b介导的脱靶效应将通过DNA突变和 RNA-seq分析。为了定义miR-29b在逆转表型中的作用，红细胞祖细胞 将评估在缺氧条件下的病。 Sub-aim B将建立最佳剂量 MiR-29b使用临床前城镇介导γ-球蛋白重新激活和HBF诱导 SCD鼠标模型。这项研究突出了一种新型基于miRNA的表观遗传学方法来诱导 HBF影响发现新药以扩大SCD的治疗选择。