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 该 SHINE II 应用程序旨在探索控制珠蛋白基因的表观遗传机制 该项目的中心假设是 miR-29b 在红细胞生成过程中的调节。 逆转 γ-珠蛋白至 β-珠蛋白基因开关并通过 DNA 调节诱导 HbF 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可有效诱导 HbF 的药物 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 然而，DNMT 抑制剂可以导致近端 γ-珠蛋白启动子 DNA 低甲基化。 产生脱靶副作用的小非编码 microRNA (miR) 是有吸引力的分子。 靶向 γ-珠蛋白基因表达的阻遏物，研究表明 miR-29b 抑制 DNA 通过直接靶向 DNMT3A 和 DNMT3B 的 3' 非翻译区进行甲基化。 该提案的目的是测试 miR-29b 作为 HbF 诱导剂的功效。 显示 miR-29b 对于重新激活 γ-球蛋白转录和 HbF 表达非常重要 靶向 MYB，一种已知的 γ-珠蛋白抑制因子，参与介导 DNA 甲基化和基因 为了检验我们的中心假设，我们将实现一个特定的目标来确定 miR-29b 介导 HBB 基因座 DNA 甲基化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。 使用体外原液探索 miR-29b 对 γ-珠蛋白调节的分子效应 从分离的外周血单核细胞中产生红系祖细胞的培养系统 这些研究结果将与正常红系祖细胞进行比较以进行验证。 miR-29b 通过近端启动子 DNA 重新激活 γ-球蛋白转录的能力 将通过 DNA 突变和甲基化来研究 miR-29b 介导的脱靶效应。 RNA-seq 分析确定 miR-29b 在逆转红系祖细胞表型中的作用。 将评估缺氧条件下的镰状细胞，子目标 B 将确定最佳剂量。 使用临床前 Townes 介导体内 γ-球蛋白再激活和 HbF 诱导的 miR-29b 本研究重点介绍了一种基于 miRNA 的新型表观遗传方法来诱导 SCD 小鼠模型。 HbF 影响新药的发现，以扩大 SCD 的治疗选择。