Defining the mechanisms of hemoglobin switching and genotoxicities associated with its manipulation

项目摘要 通过基因组编辑诱导胎儿血红蛋白（HbF，α2γ2）是一种有前途的β- 血红蛋白病我的工作重点是更好地了解γ-珠蛋白的发育调控 表达，并研究与CD 34+造血干细胞的基因组编辑相关的遗传毒性， 祖细胞（HSPC）来治疗性地诱导HbF。我最近的研究利用功能基因组学， 确定γ-珠蛋白启动子中对基因表达至关重要的关键DNA调控基序， 治疗性基因组编辑或胎儿血红蛋白的非缺失遗传性持续性（HPFH）。HPFH是一个 一种良性遗传病，点突变或小缺失导致成人持续γ-珠蛋白表达 红细胞然而，γ-珠蛋白表达的调节在正常情况下，以及在某些形式的HPFH中， 不完全定义。在平行的相关研究中，我已经在HSPC中表明，Cas9诱导的双链 由治疗性基因组编辑引起的诱导HbF的DNA断裂（DSB）可引起染色体畸变。 细胞分裂过程中的分离错误，导致微核形成和拷贝数异常。 端粒染色体片段大多数具有这些异常的细胞应该被内源性DNA消除 破坏监测机制。然而，DSB引起的微核也可导致稳定的 染色体重排、染色体断裂和恶性转化。因此，重要的是确定 这些异常是否在编辑HSPC后持续存在。对于这个K 01提案，我将继续我的两个单独的 但相关的研究线，以更好地了解γ-珠蛋白转录的调节和遗传毒性， 与治疗性基因组编辑相关以诱导HbF。具体来说，我将绘制一个新发现的监管机构， γ-珠蛋白基因座中的元件，并定义了表观遗传变化和对缺失重要的转录因子。 HPFH是由扩展β-珠蛋白基因座的内切酶规模缺失引起的，使用群体和单个 细胞基因组学（Aim 1）。与此同时，我将调查微核和染色体异常是否持续存在 在HSPC中的DSB之后。通过全基因组测序，活细胞和固定细胞免疫荧光，我将研究 Cas9诱导的HSPC中的染色体不稳定性、结构变异和DNA损伤传感途径 长期目标是研究体内染色体异常的持续性（目的2）。的 成功完成K 01职业发展奖将为我的长期职业生涯奠定基础 目标是建立一个独立的研究计划，调查基因调控的机制， DNA损伤检测利用这些信息来改进遗传疗法。拟议的研究和 在学术环境中的培训计划将确保独立的成功之路。

Project Summary Induction of fetal hemoglobin (HbF, α2γ2) by genome editing is a promising therapeutic strategy for β- hemoglobinopathies. The focus of my work is to better understand the developmental regulation of γ-globin expression and investigate the genotoxicities associated with genome editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) to induce HbF therapeutically. My recent studies have utilized functional genomics to identify key DNA regulatory motifs in the γ-globin promoter that are essential for gene expression following therapeutic genome editing or in non-deletional hereditary persistence of fetal hemoglobin (HPFH). HPFH is a benign, genetic condition in which point mutations or small deletions cause sustained γ-globin expression in adult red blood cells. However, the regulation of γ-globin expression normally, and in some forms of HPFH, remain incompletely defined. In parallel related studies, I have shown in HSPCs that Cas9-induced double-stranded DNA breaks (DSBs) resulting from therapeutic genome editing to induce HbF can cause chromosome segregation errors during cell division, leading to micronucleus formation and copy number abnormalities of the telomeric chromosomal segment. Most cells with these abnormalities should be eliminated by endogenous DNA damage surveillance mechanisms. However, micronuclei resulting from DSBs can also lead to stable chromosomal rearrangements, chromothripsis, and malignant transformation. Hence, it is important to determine whether these abnormalities persist after editing of HSPCs. For this K01 proposal, I will continue my two separate but related lines of investigation to better understand the regulation of γ-globin transcription and the genotoxicities associated with therapeutic genome editing to induce HbF. Specifically, I will map a newly discovered regulatory element in the γ-globin locus and define the epigenetic changes and transcription factors important for deletional HPFH, which is caused by kilobase-scale deletions of the extended β-globin locus, using population and single- cell genomics (Aim 1). In parallel, I will investigate whether micronuclei and chromosomal abnormalities persist after DSBs in HSPCs. Through whole genome sequencing, live-, and fixed-cell immunofluorescence, I will study Cas9-induced chromosome instability, structural variations, and DNA damage sensing pathways in HSPCs in vitro with the long-term goal of studying the persistence of chromosomal abnormalities in vivo (Aim 2). The successful completion of this K01 career development award will form the foundation for my long-term career goal of establishing an independent research program that investigates the mechanisms of gene regulation and DNA damage sensing to leverage this information for improved genetic therapies. The proposed research and training plans within the academic environment will ensure a successful path for independence.