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. 2018 Mar 22;9:247–256. doi: 10.1016/j.omtm.2018.03.007

Figure 5.

Figure 5

Detectable Adult Hb Production at Protein Levels with β-Globin Lentiviral Transduction in CD34+ Cells and PBMCs from SCD

(A) We transduced CD34+ cells (1×SCF) and PBMCs (5×SCF) from sickle cell disease (SCD) patients with lentiviral vectors encoding βT87Q-globin, β-globin, and GFP in IMDM-based serum-free differentiation media with 20% KSR. (B and C) We evaluated erythroid differentiation (GPA and CD71 expression), transduction (GFP positive), and cell counts 17 and 15 days after differentiation of (B) CD34+ cells and (C) PBMCs, respectively. (D and E) We evaluated vector copy number per cell (VCN) for both (D) CD34+ cell-derived erythroid cells and (E) PBMC-derived erythroid cells. Therapeutic adult Hb production was evaluated by Hb electrophoresis in differentiated erythroid cells with βT87Q-globin and β-globin transduction. (F and G) The βT87Q-globin or β-globin (adult Hb) production was confirmed by RP-HPLC in SCD (F) CD34+ cell- and (G) PBMC-derived erythroid cells with each β-globin transduction (p < 0.01). HbS, sickle hemoglobin; bars: mean ± SEM. All experiments except Hb electrophoresis (single run) were performed in triplicate.