A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease

Abstract

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor’s blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. DisclosuresNo relevant conflicts of interest to declare.