Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia

Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED (Direct repeat erythroid-definitive). DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene. In this study, lipofectaminTM2000 was used to transfect siRNA molecules and pSV-β-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels. Our findings showed that K562 cells were transfected by 40% using LipofectamineTM 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times. Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation; first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression.