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: 10427985
• 负责人: Phillip A Doerfler
• 金额: $ 13.08万
• 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/10427985
• 关键词: Address; Adult; Aneuploidy; Benign; Binding; Biological Assay; CD34 gene; Cell division; Cells; Chromosomal Instability; Chromosomal Rearrangement; 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; Investigational Therapies; 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; Pharmacology; Point Mutation; Population; Process; Protocols documentation; Regulation; Regulatory Element; Research; Research Training; Risk; Safety; Severities; Sickle Cell Trait; Switch Genes; TP53 gene; Thalassemia; Therapeutic; Variant; Work; beta Globin; beta Thalassemia; career; chromosome loss; chromothripsis; clinically relevant; evidence base; experimental study; functional genomics; gamma Globin; gene therapy; genome editing; genome sequencing; genotoxicity; improved; in vivo; insight; micronucleus; novel; novel therapeutic intervention; 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.

项目总结