Therapeutic levels of fetal hemoglobin in erythroid progeny of β-thalassemic CD34+ cells after lentiviral vector-mediated gene transfer

beta-Thalassemia major results from severely reduced or absent expression of the beta-chain of adult hemoglobin (alpha(2)beta(2); HbA). Increased levels of fetal hemoglobin (alpha(2)gamma(2); HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of beta-thalassemia, raising the potential for genetic therapy directed at enhancing HbF. We used an in vitro model of human erythropoiesis to assay for enhanced production of HbF after gene delivery into CD34(+) cells obtained from mobilized peripheral blood of normal adults or steady-state bone marrow from patients with beta-thalassemia major. Lentiviral vectors encoding (1) a human gamma-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the gamma-globin gene promoters, or (3) a short-hairpin RNA targeting the gamma-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34(+) cells demonstrated levels of HbF up to 21% per vector copy. For beta-thalassemic CD34(+) cells, similar gene transfer efficiencies achieved HbF production ranging from 45% to 60%, resulting in up to a 3-fold increase in the total cellular Hb content. These observations suggest that both lentiviral-mediated gamma-globin gene addition and genetic reactivation of endogenous gamma-globin genes have potential to provide therapeutic HbF levels to patients with gamma-globin deficiency. (Blood. 2011; 117(10): 2817-2826)