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

• Published in: Molecular therapy. Methods & clinical development Vol. 12; pp. 175 - 183
• Main Authors: Lux, Christopher T., Pattabhi, Sowmya, Berger, Mason, Nourigat, Cynthia, Flowers, David A., Negre, Olivier, Humbert, Olivier, Yang, Julia G., Lee, Calvin, Jacoby, Kyle, Bernstein, Irwin, Kiem, Hans-Peter, Scharenberg, Andrew, Rawlings, David J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Limited 15.03.2019; American Society of Gene & Cell Therapy; Elsevier
• Subjects: Binding sites; CD34 antigen; Cell differentiation; Clonal deletion; Deoxyribonucleic acid; Design; Disease; DNA; Fetuses; Flow cytometry; Gene deletion; Gene silencing; Genome editing; Genotype & phenotype; Hematopoietic stem cells; Hemoglobin; High-performance liquid chromatography; Medical research; Mutation; Phenotypes; Promoters; Proteins; Sickle cell disease; Stem cell transplantation; Stem cells; Thalassemia; Transcription activator-like effector nucleases; Transcription factors; Transfection; Transplants & implants; thalassemia; SCD; TALEN; HSC; HPFH; HBG; gene editing; hemoglobinopathy; hemoglobin; sickle cell disease
• ISSN: 2329-0501; 2329-0501
• DOI: 10.1016/j.omtm.2018.12.008

Elements within the γ-hemoglobin promoters ( and ) function to bind transcription complexes that mediate repression of fetal hemoglobin expression. Sickle cell disease (SCD) subjects with a 13-bp deletion in the promoter exhibit a clinically favorable hereditary persistence of fetal hemoglobin (HPFH) phenotype. We developed TALENs targeting the homologous promoters to de-repress fetal hemoglobin. Transfection of human CD34 cells with TALEN mRNA resulted in indel generation in (43%) and (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 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 and , suggesting that this approach can provide therapeutic benefit in patients with SCD or β-thalassemia.