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

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

抽象的镰状细胞病 (SCD) 和某些类型的 β 地中海贫血是由细胞缺陷引起的成人形式的血红蛋白在出生后不久就会出现，此时从胎儿的血红蛋白转变为成人形式的血红蛋白。到成人形式的血红蛋白就完成了。即使这个开关的部分逆转也是如此与这些疾病的病程改善有关。我们采用了新改进的 CRISPR-Cas9平台进行以激酶结构域为重点的遗传筛选以鉴定抑制胎儿血红蛋白（HbF）产生的潜在药物分子。这筛选发现 HRI（也称为 EIF2AK1），一种红细胞特异性蛋白激酶调节蛋白质翻译。 HRI 的消耗会提高人体 HbF 水平红系细胞几乎没有额外的扰动。 HRI 损失降低了主要 HbF 阻遏物 BCL11A，并且 BCL11A 表达恢复部分恢复 HbF 抑制。此外，HRI 消耗减少了 SCD 衍生人类的镰状化培养中的红细胞。在目标 1 中，我们将通过以下方式全面剖析 HRI 功能：评估 HRI 耗尽细胞的转录组和蛋白质组。目标 2 的目标是研究 HRI 调节 BCL11A 的机制，确定其他 HRI 调节 HbF 阻遏物，并检查 HRI 对蛋白质翻译的整体影响原代人红细胞的控制。目标 3 将探索与之前的协同效应已知的 HbF 诱导剂均使用候选方法和无偏见的基因筛选以获得新颖的协同效应。在目标 4 中，我们将通过以下方式检查 HRI 损失对 SCD 的影响：生成 HRI 缺陷的人源化 SCD 小鼠模型。总之，这些研究探索 HRI 在人类红细胞生物学中的作用并检查 HRI 作为药理学靶点单独诱导 HbF 或与机械上不同的 HbF 诱导剂联合诱导。