559. Induction of Fetal Hemoglobin in Adult Erythroblasts by Genome Editing of the Beta-Globin Locus

Abstract

Sickle cell disease (SCD) and β-thalassemia are severe anemias characterized by abnormal or reduced production of hemoglobin β-chains. Autologous transplantation of genetically corrected hematopoietic stem cells (HSC) is an attractive therapeutic alternative for patients lacking a compatible HSC donor. Naturally occurring, large deletions in the β-globin locus result in increased fetal hemoglobin (HbF) expression (HPFH, Hereditary Persistence of Fetal Hemoglobin), a condition that mitigates the clinical severity of β-hemoglobinopathies. Here, we integrated BCL11A and GATA1 transcription factor binding site analysis and HPFH mutational data to identify potential HbF silencers in the β-globin locus. Based on this analysis, we designed a CRISPR/Cas9 strategy to disrupt a 13-kb genomic region commonly deleted in HPFH, which includes the Δ- and β-globin genes and putative intergenic HbF silencers, and achieved efficient targeted deletion in erythroid cell lines by plasmid, RNA and lentiviral delivery of the CRISPR/Cas9 nuclease system. RT-PCR showed a dramatic increase in γ-globin mRNA levels in modified adult hematopoietic stem progenitor cells (HSPC)-derived erythroid cell lines (HUDEP-2). FACS and HPLC analysis demonstrated reactivation of HbF and a concomitant decrease in HbA expression. Cell morphology, erythroid marker profile, total Hb levels and erythroid maturation were unaffected, consistent with the asymptomatic phenotype of adult HPFH carriers. The same strategy was tested in primary human erythroblasts by lentiviral transduction of adult CD34+ HSPCs followed by in vitro erythroid differentiation in liquid and clonogenic cultures. Deletion of potential HbF silencers resulted in a 35% increase in γ-globin expression compared to basal levels in primary human erythroblasts as measured by HPLC, suggesting that these sequences could serve as targets for therapeutic genome editing for HbF induction in β-hemoglobinopathies. We are currently testing the efficiency of our CRISPR/Cas9-based strategy in patient-derived HSPCs. Overall, this study contributes to the knowledge of the mechanisms underlying fetal to adult Hb switching, and provides clues for a therapeutic strategy for SCD and β-thalassemia.