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

定义血红蛋白转换的机制和与其操作相关的遗传毒性

基本信息

批准号：
10579331
负责人：
Phillip A Doerfler
金额：
$ 12.93万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-04-01 至 2026-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10579331
关键词：
Address Adult Aneuploidy Benign Binding Biological Assay CD34 gene Cell division Cells Chromosomal Instability Chromosomal Rearrangement Chromosomal Stability Chromosome Segregation Chromosome Structures Chromosome abnormality Clinical Clinical Research Collaborations Cytology DNA DNA Damage DNA Double Strand Break Development Developmental Gene Distal Distant Elements Ensure Environment Epigenetic Process Erythrocytes Erythroid Cells Essential Genes Fetal Hemoglobin Foundations Gene Activation Gene Expression Gene Expression Regulation Genes Genetic Diseases Genetic Transcription Genomics Goals Hematopoietic stem cells Hemoglobin Hemoglobin F Disease Hemoglobinopathies Human Immunofluorescence Immunologic In Vitro Intercistronic Region Investigation K-Series Research Career Programs Lead Longevity Malignant - descriptor Maps Mediating Mendelian disorder Molecular Mutagenesis Mutation Nuclear Structure Nucleic Acid Regulatory Sequences Pathway interactions Perinatal Point Mutation Population Process Protocols documentation Regulation Regulatory Element Research Risk Safety Severities Sickle Cell Trait Switch Genes TP53 gene Thalassemia Therapeutic Training Variant Work beta Globin beta Thalassemia career cell fixing chromosome loss chromothripsis clinically relevant derepression evidence base experimental study functional genomics gamma Globin gene therapy genome editing genome sequencing genotoxicity improved in vivo insight micronucleus novel novel therapeutic intervention pharmacologic postnatal precision medicine preclinical study premalignant programs promoter success therapeutic genome editing therapeutic target transcription factor whole genome

项目摘要

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.

项目概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- 治疗策略我的工作重点是更好地了解 γ-珠蛋白的发育调节。表达并研究与 CD34+ 造血干细胞基因组编辑相关的遗传毒性我最近的研究利用功能基因组学来诱导 HbF。鉴定 γ-珠蛋白启动子中的关键 DNA 调控基序，这些基序对于以下基因表达至关重要治疗性基因组编辑或胎儿血红蛋白的非缺失遗传性持久性（HPFH）是一种治疗性基因组编辑。良性遗传病，其中点突变或小缺失导致成人持续表达 γ-珠蛋白然而，γ-珠蛋白表达的调节在正常情况下以及在某些形式的 HPFH 中仍然存在。在并行的相关研究中，我已经在 HSPC 中证明了 Cas9 诱导的双链。诱导 HbF 的治疗性基因组编辑导致的 DNA 断裂 (DSB) 可能导致染色体细胞分裂过程中的分离错误，导致微核形成和拷贝数异常大多数具有这些异常的细胞应被内源性 DNA 消除。然而，DSB 产生的微核也可以导致稳定。因此，确定染色体重排、染色体碎裂和恶性转化非常重要。对于这个 K01 提案，这些异常在编辑 HSPC 后是否仍然存在，我将继续我的两个单独的工作。但相关的研究路线可以更好地了解γ-珠蛋白转录的调节和遗传毒性具体来说，我将绘制一个新发现的调控图谱。 γ-球蛋白基因座中的元件，并定义对删除重要的表观遗传变化和转录因子 HPFH，是由扩展 β-珠蛋白基因座的千碱基级缺失引起的，使用群体和单同时，我将研究微核和染色体异常是否持续存在。在 HSPC 中的 DSB 之后，我将通过全基因组测序、活细胞和固定细胞免疫荧光进行研究。 Cas9 诱导的 HSPC 染色体不稳定性、结构变异和 DNA 损伤传感通路体外试验，长期目标是研究体内染色体异常的持续性（目标 2）。成功完成K01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来研究基因调控机制和 DNA 损伤传感利用这些信息来改进基因疗法。学术环境中的培训计划将确保成功的独立之路。