Lentiviral Vector-Mediated Transfer of the Gamma-Globin Gene Into Normal and Beta-Thalassemic Human CD34⁺ Cells Results in Potentially Therapeutic Levels of Fetal Hemoglobin in Erythroid Progeny.

Abstract 3579

Poster Board III-516

Beta-thalassemia results from severely reduced or absent expression of the beta-globin chain of hemoglobin, leading to severe anemia which is dependent upon transfusion for survival. The pathophysiology centers on the alpha- to beta-globin chain imbalance, which results in precipitation of excess alpha chains. These precipitates caused oxidative stress, membrane damage, and apoptosis of erythroid precursors in the bone marrow (BM). Allogeneic stem cell transplant can be curative but is only available to individuals with a matched donor. It is well known that increased levels of fetal hemoglobin (α₂γ₂;HbF), such as in the case of hereditary persistence of fetal hemoglobin, ameliorate the clinical severity of beta-thalassemia. We therefore developed a self-inactivating (SIN), erythroid-specific, gamma-globin lentiviral vector with the goal of reducing the chain imbalance through the production of HbF in red blood cells following transduction and transplantation of autologous HSCs. The vector, termed V5m3, contains 3.1-kb of transcriptional regulatory sequences from the β-globin locus control region, a 130-bp beta-globin promoter regulating transcription of the gamma-globin gene, and 3′ untranslated sequences from the native beta-globin gene. We previously showed that this vector was effective in correcting mouse models of beta-thalassemia and sickle cell disease (SCD). We further modified V5m3 to include a 400-bp chicken HS4 insulator element. Mobilized peripheral blood (PB)- or steady state bone marrow (BM)-derived CD34⁺ cells from normal or thalassemic human donors were used to evaluate vector performance. A two-phase in vitro model of human erythropoiesis was used in which a seed population of CD34⁺ cells is first expanded and then differentiated into late stage erythroblasts over a two week period. Gene transfer is performed 48 hours after initiation of culture. In this model, we routinely observe a 1000-fold expansion in total cell numbers where the vast majority of maturing erythroid cells are late stage erythroblasts reflected by nearly complete enrichment for expression of transferrin receptor (CD71; ≥98%) and glycophorin A (CD235; ≥80%) and loss or reduced expression of CD34 and CD45. Erythroid cells generated from normal adult PB and BM CD34⁺ cells demonstrate an adult pattern of hemoglobin production (HbA>96%;HbF<2%), as measured by acetate gel electrophoresis and HPLC, making this model ideal to evaluate enhancement of HbF levels by gene transfer. Normal PB CD34⁺ cells from four independent donors were transduced with the gamma-globin vector (MOI=20) or a GFP control vector (MOI=5). Vector transduction had no effect on cell growth or differentiation as monitored by consistent increases in total cell numbers and the appearance of CD71 (transferrin receptor) and glycophorin A on most cells (≥98% and '80%, respectively). The GFP vector achieved an average transduction rate of 87+/−6% and erythroblasts expressed low levels of HbF (1.7+/−0.6). Gamma globin gene transfer with the V5m3 (N=2) and V5m3-400 (N=4) vectors resulted in HbF levels ranging from 6 to 25%, with an average vector copy number of 0.8 to 1.1. We next tested the V5m3-400 vector using BM CD34⁺ cells from two patients with beta-thalassemia major. High levels of gene transfer were obtained with both the GFP and the globin vector, as evidenced by bulk marking (74+/−6% GFP+) or PCR analysis of CFU for presence of the V5m3-400 vector (12/12 positive Exp 1); (18/20 Exp 2); (18/24 Exp 3). Again, gene transfer did not perturb erythroid differentiation as monitored by the appearance glycophorin A (88+/−9%; GFP control and 86+/−6%; V5m3-400) as well as cell morphology. Erythroblasts derived from GFP transduced cells had a mean HbF level of 26+/−5% whereas those derived from cells transduced with V5m3-400 demonstrated a 100% increase of HbF to 58+/−2% with an average vector copy number of 0.6-0.8. Importantly, cultures of cells transduced with the globin vector demonstrated an average of 30% reduction in apoptotic cells (10% tunnel+) cells compared to GFP transduced control cells (15% tunnel+), suggesting rescue of erythroblasts through correction of the globin chain imbalance. Our data show that potentially therapeutic levels of HbF in thalassemic erythroblasts can be obtained following gene transfer using a gamma-globin lentiviral vector.