Inhibition of γ/β Globin Gene Switching in CD 34 +  Derived Erythroid Cells by BCL11A RNA Silencing

In this study, the possibility of increasing HbF in the CD34+ derived erythroid cells was investigated by BCL11A inhibition using specific small-interfering RNAs (siRNAs). Human peripheral blood-derived hematopoietic stem cells were isolated and differentiated to erythroid cells. Erythroid maturation was investigated using cell morphology parameters and flow cytometry analysis of CD235a expression On day 20, siRNA complementary to BCL11A was transfected to differentiating cells via electroporation. BCL11A expression was evaluated through real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbant assay (ELISA). β actin was used as the reference gene to confirm the relative expression level of BCL11A gene mRNA. 48 hours after transfection, BCL11A siRNA significantly reduced BCL11A mRNA levels and consequently led to 2.0 fold decrease in corresponding protein. On the 28th day, haemoglobin electrophoresis re sults showed that Hb F levels in transfected erythroid cells increased 3.3-fold when compared with non transfected cells. In this study we showed that BCL11A inhibition in erythroid cells could increase fetal hemoglobin, and this strategy can be the basis for designing a γ globin expressing cellula r system to increase Hb F in patients with thalassemia and SCD.
Source: Indian Journal of Hematology and Blood Transfusion - Category: Hematology Source Type: research