βT87Q-globin gene therapy reduces sickle hemoglobin production, allowing for ex vivo anti-sickling activity in human erythroid cells

Abstract 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 (s) HUDEP-2 cells produced robust HbS after differentiation and sickled under deoxygenated conditions, comparable to SCD CD34+ progeny. Lentiviral transduction provided 9.5-26.8 pg/cell of βT87Q-globin (R2=0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R2=0.92). Interestingly, βT87Q-globin transduction markedly reduced endogeneous βS-globin (R2=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-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/βS-globin suppression. In summary, using sHUDEP-2 and CD34+ derived cells, we demonstrated that lentiviral addition of βT87Q-globin strongly reduced endogeneous β-/βS-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.