TALEN-Mediated Gene Editing of HBG in Human Hematopoietic Stem Cells Leads to Therapeutic Fetal Hemoglobin Induction

Abstract

Elements within the γ-hemoglobin promoters (HBG1 and HBG2) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the HBG1 promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous HBG promoters to de-repress fetal hemoglobin. Transfection of human CD34+ cells with TALEN mRNA resulted in indel generation in HBG1 (43%) and HBG2 (74%) including the 13-bp HPFH deletion (∼6%). Erythroid differentiation of edited cells revealed a 4.6-fold increase in γ-hemoglobin expression as detected by HPLC. Assessment of TALEN-edited CD34+ cells in vivo in a humanized mouse model demonstrated sustained presence of indels in hematopoietic cells up to 24 weeks. Indel rates remained unchanged following secondary transplantation consistent with editing of long-term repopulating stem cells (LT-HSCs). Human γ-hemoglobin expressing F cells were detected by flow cytometry approximately 50% more frequently in edited animals compared to mock. Together, these findings demonstrate that TALEN-mediated indel generation in the γ-hemoglobin promoter leads to high levels of fetal hemoglobin expression in vitro and in vivo, suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia. Keywords: TALEN, hemoglobinopathy, HBG, hemoglobin, HPFH, SCD, sickle cell disease, thalassemia, gene editing, HSC