A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Erythrocytes Sickling in Human Erythroid Culture Model

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, 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 substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34⁺ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34⁺ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34⁺ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation.