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

Abstract

β-Thalassemia major results from severely reduced or absent expression of the β-chain of adult hemoglobin (α2β2;HbA). Increased levels of fetal hemoglobin (α2γ2;HbF), such as occurs with hereditary persistence of HbF, ameliorate the severity of β-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 β-thalassemia major. Lentiviral vectors encoding (1) a human γ-globin gene with or without an insulator, (2) a synthetic zinc-finger transcription factor designed to interact with the γ-globin gene promoters, or (3) a short-hairpin RNA targeting the γ-globin gene repressor, BCL11A, were tested. Erythroid progeny of normal CD34+ cells demonstrated levels of HbF up to 21% per vector copy. For β-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 γ-globin gene addition and genetic reactivation of endogenous γ-globin genes have potential to provide therapeutic HbF levels to patients with β-globin deficiency.

The New Self-Inactivating Lentiviral Vector for Thalassemia Gene Therapy Combining Two HPFH Activating Elements Corrects Human Thalassemic Hematopoietic Stem Cells

“…On the basis of the above-described findings, by employing this culture system, we were able to reduce the in vitro percentage of HbF produced from thalassemic samples relative to the in vivo values, a strategy that has been successfully used in assessments of HbF production via gene transfer approaches (Wilber et al, 2011). As shown in Fig.…”

“…For that reason, we first focused on the use of human primitive hematopoietic cells from various developmental stages to derive erythroid cultures of maturing erythroblasts that can be used to efficiently evaluate the molecular mechanisms of switching. In addition, we established a culture system with minimal production of HbF in vitro, a feature that permits accurate assessment of vector-derived c-globin production as previously described (Wilber et al, 2011). This culture system actually resulted in low levels of HbF production in cells derived from adult bone marrow and in high levels of HbF in cells derived from cord blood, suggesting that it represents a reliable recapitulation of the in vivo status.…”

“…Color images available online at www.liebertonline.com/hum reported globin insulated vectors (Puthenveetil et al, 2004) exhibited low titers and had to be concentrated 10,000-fold, probably because of the coexistence of parts of the LCR and the 1.2-kb cHS4 in the 3¢ LTR that altogether result in a rather large genomic size for packaging into a lentivirus. To address this specific point, we proceeded to a head-to-head comparison of the titer obtained both by GGHI and by vector V5m3-400, which is another c-globin lentiviral vector that comprises the LCR region and a 400-bp fragment of the cHS4 insulator within the U3 region (Wilber et al, 2011). In accordance with procedures done in parallel for both vectors, the generated titers were 2 · 10 8 TU/ml for GGHI and 1 · 10 9 TU/ml for V5m3-400.…”

“…Because in our case a large insertion in the U3 region had no apparent effect on the vector's stability and titer, it is conceivable that the combination of both insulators incorporated in the U3 region along with large expression cassettes (e.g., LCR-cassettes) could be the probable cause for the reported LCR-vector titer reduction (Puthenveetil et al, 2004). To clarify this issue, we made a head-to-head comparison of the titers obtained both by GGHI and by another c-globin lentiviral vector designated V5m3-400, that comprises the LCR region and a 400-bp fragment of the cHS4 insulator within the U3 region (Wilber et al, 2011). For these specific experiments, the V5m3-400 vector batches were made in exactly the same way as the GGHI vector batches and the titration procedures were performed in parallel for both vectors.…”

Thalassemia and its Management during Pregnancy

“…One participant in an ongoing clinical trial has achieved independence from transfusion after gene transfer into bone marrow stem cells mediated via lentivirus. 72 Ongoing efforts need to be focused on improving the efficiency of lentiviral vector-mediated gene transfer into stem cells so that the curative potential of gene transfer can be consistently achieved.…”