Figure 4.

Testing of LV AS3m.13bpdel and LV AS3m.BCL11A in SCD HSPCs

(A) Schematic representation of the protocol used to transduce and transfect SCD HSPCs. 24 h after thawing, HSPCs were transduced with LV AS3m.13bpdel and LV AS3m.BCL11A, and after 48 h cells were transfected with the Cas9-GFP protein and differentiated into mature RBCs following a 21-day differentiation protocol. (B) Correlation between VCN and indel frequency in cells treated with LVs AS3m.BCL11A (one mobilized SCD donor) and AS3m.13bpdel (two mobilized SCD donors). R² and line-of-best-fit equation are indicated. (C) Deep-sequencing analyses of the 13bpdel off-target in mature erythroblasts derived from adult SCD HSPCs treated with LV AS3m.13bpdel. Transduced and mock-transfected SCD cells (Ctr) are indicated in red and edited samples (AS3m.13bpdel) in black. (D) GUIDE-seq analysis of gR-BCL11A in 293T cells. The protospacer targeted by gR-BCL11A and the PAMs are shown in the first line. Off-targets and their mismatches with the on-target (highlighted in color), sequencing read counts, and chromosomal position are reported. (E) Relative expression of HBG mRNA normalized to α-globin in untreated SCD cells (Ctr) (n = 5) and cells treated with LV AS3m.13bpdel (n = 14, 2 SCD donors). ∗∗∗p < 0.001 (unpaired t test). Horizontal lines indicate median and first and third quartiles. (F) Representative flow cytometry analysis of F cells in RBCs derived from untreated (Ctr UT; light blue), and LV AS3m.13bpdel-treated SCD HSPCs mock transfected (medium blue) or transfected with Cas9-GFP (dark blue) (two mobilized SCD donors). (G) Correlation between VCN and percentage of HbS and HbAS3 (determined by CE-HPLC) in cells derived from mock-transfected (orange and light blue) and Cas9-transfected (red and dark blue) SCD HSPCs transduced with LV AS3m.13bpdel (two mobilized SCD donors). We plotted the percentage of HbS or HbAS3+HbF over the total Hbs. R² and line-of-best-fit equation are indicated. Dashed lines indicate the VCN required to achieve equal amounts of HbS and HbAS3. (H) Correlation between indel frequency and percentage of HbS and HbAS3 (determined by CE-HPLC) in cells derived from Cas9-transfected SCD HSPCs transduced with LV AS3m.13bpdel (two mobilized SCD donors). R² and line-of-best-fit equation are indicated.