DEGRADATION OF BCL11A PROTEIN FOR HbF REACTIVATION

项目总结/摘要 在成人红系细胞中重新激活胎儿血红蛋白表达是一种有效的遗传学方法。 b-地中海贫血和镰状细胞病（SCD）的治疗。然而，按照目前的做法， 基因治疗（慢病毒递送或CRISPR/Cas9编辑）无法满足大规模疾病 由于其依赖于患者的清髓性预处理， 重组和程序的医疗密集性。药物（小分子） 需要治疗剂来治疗患有这些疾病的许多患者。我们的研究愿景 是使用小分子疗法来重新激活γ-珠蛋白基因的表达， 而且安全最有效的，经验证的g-珠蛋白表达的阻遏物是BCL 11 A。在这个项目中 我们将扩展我们最近的研究，在这些研究中，我们利用新的化学方法， 在一些实施方案中，通过TPD降解BCL 11 A以消耗BCL 11 A并重新激活HbF产生。该项目包括两个 广泛的目标。首先，我们将使用一个平台来确定珠蛋白基因转录的动力学， 处于细胞周期不同阶段的红系细胞可被分离用于新生转录 分析。使用BCL 11 A的急性TPD，我们将确定在细胞周期的哪个阶段g- (HBG)球蛋白在失去阻遏物后被重新激活。这些数据将提供时间分辨率 以前所未有的分辨率记录了珠蛋白基因的转录。在第二个目标中，我们将探索使用 红系特异性E3泛素连接酶TRIM 10和TRIM 58靶向降解BCL 11 A。的 TRIM蛋白将被导向至具有BCL 11 A特异性纳米抗体的BCL 11 A。我们将启动发现程序 以开发用于BCL 11 A的细胞特异性TPD的工具化合物 作为一种新的治疗策略。我们的工作将为新的小分子方法奠定基础 用于血红蛋白紊乱的HbF的稳健和安全的再激活。

Project Summary/Abstract Reactivation of fetal hemoglobin expression in adult erythroid cells is a validated approach to genetic therapy of both b-thalassemia and sickle cell disease (SCD). As currently practiced, however, genetic therapy (either lentiviral delivery or CRISPR/Cas9 editing) cannot meet the large disease burden due to its reliance on myeloablative preconditioning of patients for hematopoietic reconstitution and the medically intensive nature of the procedure. Drug (small molecule) therapeutics are needed to treat the many patients with these disorders. The vision of our research is to use small molecule therapeutics to reactivate y-globin gene expression, and do so both robustly and safely. The most potent, validated repressor of g-globin expression is BCL11A. In this project we will extend our recent studies in which we leveraged new chemical methods of targeted protein degradation (TPD) to deplete BCL11A and reactivate HbF production. The project consists of two broad aims. First, we will determine the dynamics of globin gene transcription using a platform in which erythroid cells at different phases of the cell cycle can be isolated for nascent transcription analyses. Using acute TPD of BCL11A, we will then ascertain at which stage(s) of the cell cycle g- (HBG) globin is reactivated upon loss of the repressor. These data will provide temporal resolution of globin gene transcription at unprecedented resolution. In a second aim, we will explore the use of erythroid-specific E3 ubiquitin ligases, TRIM10 and TRIM58, to target degradation of BCL11A. The TRIM proteins will be directed to BCL11A with BCL11A-specific nanobodies. We will initiate discovery of ligands for the TRIM proteins in order to develop tool compounds for cell-specific TPD of BCL11A as a new therapeutic strategy. Our work will lay the groundwork for novel small molecule approaches for robust and safe reactivation of HbF for the hemoglobin disorders.