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

• 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
• 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

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.

项目摘要 通过基因组编辑诱导胎儿血红蛋白（HBF，α2γ2）是β-的有前途的治疗策略 血红蛋白病。我工作的重点是更好地了解γ-球蛋白的发展调节 表达并研究与CD34+造血茎的基因组编辑相关的遗传毒性和 祖细胞（HSPC）热诱导HBF。我最近的研究将功能基因组学用于 鉴定γ-珠蛋白启动子中的关键DNA调节基序，这对于基因表达至关重要 治疗性基因组编辑或胎儿血红蛋白（HPFH）的非缺血遗传持久性。 HPFH是一个 在成年 红细胞。然而，正常对γ-球蛋白表达的调节，并以某些形式的HPFH保持 未完全定义。在并行相关的研究中，我在HSPC中表明CAS9诱导的双链 热基因组编辑引起的DNA断裂（DSB）诱导HBF会引起染色体 细胞分裂期间的隔离错误，导致微核形成和拷贝数异常 远程染色体段。大多数具有这些异常的细胞应通过内源性DNA消除 损坏监视机制。但是，由DSB产生的微核也可能导致稳定 染色体重排，染色体和恶性转化。因此，确定很重要 这些异常是否在编辑HSPC后持续存在。对于此K01提案，我将继续我的两个单独 但是相关的投资行，以更好地了解γ-珠蛋白转录和遗传毒素的调节 与热基因组编辑相关，以诱导HBF。具体来说，我将绘制一个新发现的监管 γ-珠蛋白基因座的元素并定义了对缺失重要的表观遗传变化和转录因子 HPFH是由扩展的β-珠蛋白基因座的千目标尺度缺失引起的，使用了种群和单一 细胞基因组学（AIM 1）。同时，我将研究微核和染色体异常是否持续 在HSPC中的DSB之后。通过整个基因组测序，活和固定细胞免疫荧光，我将研究 Cas9引起的染色体不稳定性，结构变化和HSPC中的DNA损伤敏感性途径 体外的长期目标是研究体内染色体异常的持续性（AIM 2）。这 成功完成这个K01职业发展奖将构成我长期职业的基础 建立独立研究计划的目标，该计划研究基因调节的机制和 DNA损伤灵敏度利用此信息来改善基因疗法。拟议的研究和 在学术环境中的培训计划将确保成功实现独立性。