A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Cultured Mouse Erythrocytes

Abstract

β-hemoglobinopathies are inherited disorders caused by mutations/deletions in the β-globin chain that lead to structurally defective β-globin chains or reduced (or absent) β-globin chain production. These diseases affect multiple organs and are associated with considerable morbidity and mortality, representing a major public health challenge. Current gene therapy approaches for the 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 human CD34+ cells. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from mouse hematopoietic progenitor 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 1.8-fold, δ-globin gene expression 3.3-fold, and γ-globin gene expression 1.7-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.6-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2 was increased from 2.1 % in vector-transduced cells to 8.9% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 6.3% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 30.6% in medium-form Eklf-GATA-transduced cells as compared with 40.7% 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. ChIP-sequencing analysis has demonstrated that Eklf-GATA1 fusion proteins and GATA1 having a similar protein-DNA binding pattern at a global level. Our results have found that the long form Eklf-GATA1 fusion protein has a major effect on δ-globin induction than β-globin; the medium form Eklf-GATA1 is able to elevate δ-globin expression without having an effect on β-globin expression. The above findings 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.