Project Summary
Induction of fetal hemoglobin (HbF, a2¿2) by genome editing is a promising therapeutic strategy for ß-
hemoglobinopathies. The focus of my work is to better understand the developmental regulation of ¿-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 ¿-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 ¿-globin expression in adult
red blood cells. However, the regulation of ¿-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 ¿-globin transcription and the genotoxicities
associated with therapeutic genome editing to induce HbF. Specifically, I will map a newly discovered regulatory
element in the ¿-globin locus and define the epigenetic changes and transcription factors important for deletional
HPFH, which is caused by kilobase-scale deletions of the extended ß-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.