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

Selami Demirci; Bjorg Gudmundsdottir; Quan Li; Juan J Haro-Mora; Tina Nassehi; Claire Drysdale; Morgan Yapundich; Jackson Gamer; Fayaz Seifuddin; John F Tisdale; Naoya Uchida

doi:10.1016/j.omtm.2020.04.013

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

Mol Ther Methods Clin Dev. 2020 Apr 18:17:912-921. doi: 10.1016/j.omtm.2020.04.013. eCollection 2020 Jun 12.

Affiliations

¹ Cellular and Molecular Therapeutics Branch, National Heart Lung and Blood Institutes (NHLBI)/National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD, USA.
² Laboratory of Chemical Physics, NIDDK, NIH, Bethesda, MD, USA.
³ Bioinformatics and Computational Biology Laboratory, NHLBI, NIH, Bethesda, MD, USA.

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 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 (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 endogenous β^S-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 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.

Keywords: gene therapy; hemoglobin; immortalized erythrocytes; sickle cell disease.