Downregulation of Transcription Factor LRF/ZBTB7A Increases Fetal Hemoglobin Expression in β-Thalassemia/Hemoglobin E Erythroid Cells

LRF/ZBTB7A is a transcription factor that has been recently identified as a new key regulator of fetal hemoglobin (HbF; α2γ2) production in erythroid cells. Reduction of LRF/ZBTB7A expression led to increases in levels of HbF in human CD34+ hematopoietic stem and progenitor cell (HSPC)-derived erythroblast and in human immortalized erythroid line (HUDEP-2). Since reactivation of γ-globin gene is associated with the improvement of clinical manifestations of β-hemoglobinopathy patients, decrement in LRF/ZBTB7A expression might be a substantial interest as a novel target for gene therapy in β-thalassemia. In this study, we investigated the effects of LRF/ZBTB7A downregulation in erythroid cells derived from β-thalassemia/HbE patients in order to evaluate its therapeutic potential. The hematopoietic CD34+ progenitor cells were collected from 3 patients and 3 healthy normal individuals' peripheral blood and subjected for in vitro erythroblast culture. The cells were transduced with lentivirus carrying LRF/ZBTB7A specific shRNA, and used untransduced cells and non-targeted control shRNA (shNTC) as experimental controls. The LRF/ZBTB7A shRNA reduced LRF/ZBTB7A transcript and protein to nearly undetectable levels. Interestingly, downregulation of LRF/ZBTB7A increased expression of γ-globin, ε-globin and ζ-globin in both adult normal and β-thalassemia/HbE derived cells, whereas α-globin, β-globin and δ-globin expression were decreased. As previously reported, we found that the LRF/ZBTB7A knockdown produced a robust increase in HbF levels in both normal (43.3±9.0% vs. 5.9±2.1% in shNTC) and β-thalassemia/HbE erythroblasts (78.1±3.5% vs. 26.3±3.9% in shNTC). Noteworthy, the delay of erythroid differentiation was observed in the LRF/ZBTB7A knockdown cells of both derived from β-thalassemia/HbE patients and normal control, suggesting an additional role of LRF/ZBTB7A in regulating erythroid maturation. These data support the manipulation of LRF/ZBTB7A as one of the most interesting gene therapy candidates for treating the β-thalassemia, but the effect on erythroid cell maturation is needed to be concerned and required further investigation. Disclosures No relevant conflicts of interest to declare.