The bifunctional LV downregulating HbS does not generate a β-thalassemic phenotype
(A) Schematic representation of the protocol used to evaluate erythroid differentiation and RBC parameters. We transduced SCD/HD HSPCs with LV AS3m.C and transfected them with Cas9 protein. As controls, we transfected SCD/HD HSPCs with RNPs containing gR-C. Images of in vitro-cultured RBCs were taken using the Phasics SID4-HR GE camera. The BIO-Data interface software was used to analyze RBC images. (B) CE-HPLC quantification of Hb tetramers in untreated HD and SCD cells (Ctr UT), gR-C-treated HD and SCD cells (gR-C), and LV AS3m.C-treated SCD HSPCs that were mock transfected (“–”) or transfected with Cas9-GFP protein. We plotted the percentage of each Hb type over the total Hb tetramers. VCN values are reported in blue and indels in black below the graph. The α-globin/non-α-globin ratio (determined by RP-HPLC) is reported on top of the histograms.