Βt87q-Globin Gene Therapy Reduces Sickle Hemoglobin Production, Allowing for Ex Vivo Anti-sickling Activity in Human Erythroid Cells

Lentiviral addition of beta T87Q-globin, a modified beta-globin with an anti-sickling mutation, is currently being used in gene therapy trials for sickle cell disease (SCD) and beta-thalassemia patients. beta T87Q-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 beta T87Q-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/cellbT87Q-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, beta T87Q-globin transduction markedly reduced endogenous beta(S)-globin (R-2 = 0.84) to an undetectable level (0.4-16.8 pg/cell) in sHUDEP-2 cells, as well as endogenous beta-globin in human CD34(+) cell-derived erythroid cells. RNA sequencing (RNA-seq) analysis with beta T87Q-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 beta T87Q-globin expression and endogenous beta/beta(S)-globin suppression. In summary, using sHUDEP-2 and CD34(+)-derived cells, we demonstrated that lentiviral addition of beta T87Q-globin strongly reduced endogenous beta-/beta(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.