Functions, mechanisms, and therapeutic potential of fetal hemoglobin inducers

摘要 镰状细胞病(SCD)和某些类型的b-地中海贫血是由 成人形式的血红蛋白在出生后不久就显现出来，当胎儿从 到成人形态的血红蛋白是完整的。即使这种转变的部分逆转也是 与改善这些疾病的病程有关。我们雇用了一名新改进的 CRISPR-Cas9平台开展以激活域为重点的遗传筛查以识别 潜在的可药物分子，抑制胎儿血红蛋白(HBF)的产生。这 筛查发现HRI(也称为EIF2AK1)，一种红系特异的蛋白激酶 它调节蛋白质的翻译。HRI耗竭可提高人类HbF水平 红系细胞，几乎没有额外的扰动。HRI缺失降低了血管紧张素转换酶的表达 主要的HBF抑制因子BCL11A，BCL11A表达的恢复部分恢复 HBF压抑。此外，HRI缺失减少了SCD来源的人类的镰状症状 培养中的红系细胞。在目标1中，我们将通过以下方式全面剖析HRI的功能 评估HRI耗竭细胞的转录组和蛋白质组。目标2的目标是 要研究HRI调节BCL11A的机制，请确定其他HRI 调节HBF抑制物，并研究HRI对蛋白质翻译的全球影响 原代人类红系细胞的对照。目标3将探索与以前的协同效应 已知的HBF诱导物既使用候选方法，也通过无偏见的基因筛选 以实现新的协同效应。在目标4中，我们将通过以下方式研究HRI丢失对SCD的影响 建立HRI缺陷的人源化SCD小鼠模型。总而言之，这些研究探索了 HRI在人类红细胞生物学中的作用及其作为药理学靶点的研究 HBF诱导剂单独或与机械上不同的HBF诱导剂联合使用。

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.