Lentiviral addition of bT87Q-globin, a modified b-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and b-thalassemia patients. bT87Q-globin interferes with sickle hemoglobin (HbS) polymerization. Here, we generated the SCD mutation in an immortalized human erythroid cell line (HUDEP-2) to investigate the anti-sickling activity of bT87Q-globin. Sickle HUDEP-2 (sHUDEP-2) cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable with SCD CD34 + progeny. Lentiviral transduction provided 9.5-26.8 pg/cell bT87Q-globin (R 2 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R 2 = 0.92). Interestingly, bT87Q-globin transduction markedly reduced endogenous b S -globin (R 2 = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous b-globin in human CD34 + cellderived erythroid cells. RNA sequencing (RNA-seq) analysis with bT87Q-transduced sHUDEP-2 and human CD34 +derived cells revealed activation of inflammation-and proliferation-related programs, suggesting minimal changes in background gene expression except for bT87Q-globin expression and endogenous b/b S -globin suppression. In summary, using sHUDEP-2 and CD34 + -derived cells, we demonstrated that lentiviral addition of bT87Q-globin strongly reduced endogenous b-/b S -globin expression, resulting in an anti-sickling effect. Our findings should be helpful to understand the anti-sickling effects of therapeutic genes in SCD gene therapy.