I am studying developmental globin gene expression in order to better understand and treat sickle cell disease (SCD) and ?-thalassemia, common disorders that cause substantial morbidity and early mortality around the world. Both diseases become symptomatic between birth and 6 months age, as the ?-globin (HBG1/2) genes switch to ?-globin (HBB) causing fetal hemoglobin (HbF, ?2?2) to be replaced by adult hemoglobin (HbA, ?2?2). Elevated postnatal levels of HbF, including a benign condition termed hereditary persistence of fetal hemoglobin (HPFH), alleviates SCD and ?-thalassemia. We are seeking to reverse the ?-to-?-globin gene switch in order to induce HbF therapeutically. Our preliminary studies show that CRISPR/Cas9-mediated genome editing of primary human hematopoietic stem and progenitor cells (HPSCs) can recreate an HPFH-associated 13- nucleotide deletion in the HBG1 promoter by removing nucleotides -102 to -114 upstream of the transcriptional start site. In vitro differentiation of the gene-edited HSPCs generates erythroid progeny with HbF elevated to potentially therapeutic levels. Now, I will fine-map the -102 to -114 region using genome editing-mediated homology directed repair and cellular assays for Hb switching in order to better define the relevant nucleotide motif(s) and their interacting transcription factors (Aim 1). Because HBG1 and HBG2 are nearly identical, CRISPR/Cas9 gene editing of the -102 promoter regions introduces double-stranded DNA breaks in both genes simultaneously, which could cause deletion of the 5 kb intervening sequence, including HBG2. Preliminary studies indicate that approximately 20% of alleles harbor the 5 kb deletion after editing of human CD34+ HSPCs. Surprisingly, however, erythroid clones with this deletion exhibit increased ?-globin production, despite loss of HBG2. Epigenetic analysis and functional testing of targeted deletions within the deleted HBG1-HBG2 intergenic region identified a potential DNA regulatory element that represses HBG1 transcription. I will better define this element by mutational analysis using cellular assays for globin switching and epigenetic studies to map associated transcription factors and effects on three-dimensional chromatin structure (Aim 2). By characterizing the cis elements and the corresponding trans-acting factors that regulate ?-to-? globin switching, I hope to develop new ways to induce HbF for treating SCD and ?-thalassemia.
8. Project Narrative Hereditary persistence of fetal hemoglobin (HPFH) mutations reduce the severity of ?-hemoglobinopathies and can be introduced experimentally. We aim to study one form of HPFH in order to define the mechanisms involved in the reactivation of fetal hemoglobin. Once the mechanisms are better understood, it may be possible to develop precision medicine-based treatments for ?-hemoglobinopathies.