Functions, mechanisms, and therapeutic potential of fetal hemoglobin inducers

胎儿血红蛋白诱导剂的功能、机制和治疗潜力

• 批准号: 10308676
• 负责人: Gerd A Blobel
• 金额: $ 65.29万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-08-05 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10308676
• 关键词: 5' Untranslated Regions; Accounting; Adult; Animals; Attenuated; Biological Process; Birth; CRISPR screen; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cells; Cellular biology; Chromatin; Clone Cells; Codon Nucleotides; Collaborations; Complement; Custom; Data; Defect; Disease; Enzymes; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Fetal Hemoglobin; Foundations; Genes; Genetic Screening; Genetic Transcription; Genetic Translation; Globin; Goals; Hematopoietic stem cells; Hemoglobin; Hemoglobin concentration result; Hemoglobinopathies; Human; Knockout Mice; Laboratories; Libraries; Light; Mass Spectrum Analysis; Measures; Medical; Messenger RNA; Monitor; Mus; Open Reading Frames; PRKR gene; Panthera leo; Patients; Pharmacology; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Pre-Clinical Model; Process; Production; Protein Kinase; Proteins; Proteome; Proteomics; Regulation; Repression; Resolution; Ribosomes; Role; Severity of illness; Sickle Cell Anemia; Sum; Surveys; Testing; Thalassemia; Therapeutic; Therapeutic Index; Tissues; Translations; Western Blotting; Work; base; beta Globin; beta Thalassemia; combinatorial; design; detection limit; druggable target; experimental study; fetal; gamma Globin; improved; improved outcome; insight; mouse model; mutant; novel; restoration; sickling; small molecule; synergism; tool; transcription factor; transcriptome

Abstract Sickle cell disease (SCD) and some types of b-thalassemia that are caused by defects in the adult form of hemoglobin manifest shortly after birth, when the switch from the fetal to the adult form of hemoglobin is complete. Even a partial reversal of this switch is associated with an improved course of these diseases. We employed a newly improved CRISPR-Cas9 platform to carry out a kinase domain-focused genetic screen to identify potentially druggable molecules that repress fetal hemoglobin (HbF) production. This screen uncovered HRI (also known as EIF2AK1), an erythroid-specific protein kinase that regulates protein translation. Depletion of HRI elevates HbF levels in human erythroid cells with few additional perturbations. HRI loss reduces the expression of the major HbF repressor BCL11A, and restoration of BCL11A expression partially restores HbF repression. Moreover, HRI depletion reduces sickling of SCD-derived human erythroid cells in culture. In Aim 1 we will comprehensively dissect HRI function by assessing the transcriptome and proteome of HRI-depleted cells. The goals of Aim 2 are to study the mechanism by which HRI regulates BCL11A, identify additional HRI regulated HbF repressors, and examine the global impact of HRI on protein translational control in primary human erythroid cells. Aim 3 will explore synergies with previously known HbF inducers both using a candidate approach, and by unbiased genetic screens for novel synergies. In Aim 4 we will examine the effects of HRI loss on SCD by generating HRI-deficient humanized SCD mouse models. In sum, these studies explore the role of HRI in human red cell biology and examine HRI as target for pharmacologic HbF induction alone or in combination with mechanistically distinct HbF inducers.

摘要 镰状细胞病（SCD）和某些类型的b-地中海贫血是由缺陷引起的， 成人形式的血红蛋白在出生后不久就显现出来， 到成人血红蛋白的转变是完全的。即使是这种转换的部分逆转， 与这些疾病的改善过程有关。我们雇用了一个新改进的 CRISPR-Cas9平台开展以激酶结构域为重点的基因筛选， 抑制胎儿血红蛋白（HbF）产生的潜在药物分子。这 HRI（也称为EIF 2AK 1）是一种红细胞特异性蛋白激酶， 调节蛋白质翻译的基因HRI的消耗升高人类中的HbF水平 红系细胞，几乎没有额外的扰动。HRI损失减少了 主要HbF阻遏物BCL 11 A，BCL 11 A表达的恢复部分恢复 HbF抑制。此外，HRI消耗减少SCD衍生的人源性细胞的镰状化。 培养的红细胞。在目标1中，我们将全面剖析HRI功能， 评估HR I耗尽的细胞的转录组和蛋白质组。目标2的目标是 为了研究HRI调节BCL 11 A的机制， 调节HbF阻遏物，并检查HRI对蛋白质翻译的整体影响 在原代人红系细胞中的对照。Aim 3将探索与以前的 使用候选方法和通过无偏遗传筛选的已知HbF诱导剂 创新的协同效应。在目标4中，我们将通过以下方式检查HRI损失对SCD的影响： 产生HRI缺陷的人源化SCD小鼠模型。总之，这些研究探索 HRI在人红细胞生物学中作用及作为药理学靶点的研究 单独或与机制上不同的HbF诱导剂组合的HbF诱导。