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Lentiviral addition of βT87Q-globin, a modified β-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and β-thalassemia patients. βT87Q-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 βT87Q-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 βT87Q-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, βT87Q-globin transduction markedly reduced endogenous β S -globin (R 2  = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous β-globin in human CD34 + cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with βT87Q-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 βT87Q-globin expression and endogenous β/β S -globin suppression. In summary, using sHUDEP-2 and CD34 + -derived cells, we demonstrated that lentiviral addition of βT87Q-globin strongly reduced endogenous β-/β 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.

βT87Q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells