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

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 = 0.83) in a vector copy number (VCN)-dependent manner, resulting in a significant reduction of sickling ratios (R = 0.92). Interestingly, βT87Q-globin transduction markedly reduced endogenous β-globin (R = 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-globin suppression. In summary, using sHUDEP-2 and CD34-derived cells, we demonstrated that lentiviral addition of βT87Q-globin strongly reduced endogenous β-/β-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.