Figure 3.
Linear recruitment of the LCR reactivates fetal globin in immortalized cell lines derived from patients with SCD and HSPCs. (A) The frequency of indels, as measured by TIDE analysis, for ΔBCL11A (gRNA1617), Δ115, 500, and HS2 gRNAs in single-gRNA transfected samples in 2 SCD cell lines shown as average ± standard error of the mean (SEM). (B) Frequency of the 25-kb deletion (blue), inversion (lime green), and unmodified (gray) iteration as measured by ddPCR in Del2Rec cells. (C) RT-qPCR analysis of HBG mRNA levels in proliferating cells, displayed as the average ± SEM in percentage of the total HBB + HBG mRNA. (D) Same as in panel C but for cells differentiated for 4 days. (E) HbF of 2 Rotterdam erythroblast (REB)–SCD cell lines, as measured by HPLC, shown as the average ± SEM. It was calculated as the percentage of HbF over the total Hb tetramers. (F) Flow cytometry plots showing the percentage of HbF positive cells in 2 REB-SCD cell lines. Note that SCD1 shows relatively high baseline HbF levels. (G) Representative images of SCD cells under hypoxic conditions of a WT clone (top) and homozygous Del2Rec SCD clone (bottom). (H) HPLC analysis results of a homozygous SCD Del2Rec clone, calculated as the percentage of HbF (pink) and HbS (gray) over the total Hb tetramers. (I) Graph showing the percentage of HbF-positive cells of donors 1 to 3, as measured by flow cytometry. Error bars show the standard deviation (SD) of the triplicate samples. (J) Frequency of the 25-kb deletion (blue) and inversion (lime green) measured by ddPCR on Del2Rec transfected cells. The values were calculated as an average of 2 measurements from genomic DNA isolated from donors 4 to 6. The error bars represent the SD. (K) Frequency of indels, as measured by TIDE analysis, for ΔBCL11A (gRNA1617) of donors 4 to 6. (L) Percentage of F cells plotted as averages of 3 donors after 12 days of differentiation. A 1-way analysis of variance and Tukey’s post hoc test was used to calculated statistics. (M) HbF of donors 3 to 6 measured by HPLC 3 days after differentiation in control, ΔBCL11A, and Del2Rec conditions. We calculated the percentage of HbF over the total Hb tetramers. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; WT, wild-type.

Linear recruitment of the LCR reactivates fetal globin in immortalized cell lines derived from patients with SCD and HSPCs. (A) The frequency of indels, as measured by TIDE analysis, for ΔBCL11A (gRNA1617), Δ115, 500, and HS2 gRNAs in single-gRNA transfected samples in 2 SCD cell lines shown as average ± standard error of the mean (SEM). (B) Frequency of the 25-kb deletion (blue), inversion (lime green), and unmodified (gray) iteration as measured by ddPCR in Del2Rec cells. (C) RT-qPCR analysis of HBG mRNA levels in proliferating cells, displayed as the average ± SEM in percentage of the total HBB + HBG mRNA. (D) Same as in panel C but for cells differentiated for 4 days. (E) HbF of 2 Rotterdam erythroblast (REB)–SCD cell lines, as measured by HPLC, shown as the average ± SEM. It was calculated as the percentage of HbF over the total Hb tetramers. (F) Flow cytometry plots showing the percentage of HbF positive cells in 2 REB-SCD cell lines. Note that SCD1 shows relatively high baseline HbF levels. (G) Representative images of SCD cells under hypoxic conditions of a WT clone (top) and homozygous Del2Rec SCD clone (bottom). (H) HPLC analysis results of a homozygous SCD Del2Rec clone, calculated as the percentage of HbF (pink) and HbS (gray) over the total Hb tetramers. (I) Graph showing the percentage of HbF-positive cells of donors 1 to 3, as measured by flow cytometry. Error bars show the standard deviation (SD) of the triplicate samples. (J) Frequency of the 25-kb deletion (blue) and inversion (lime green) measured by ddPCR on Del2Rec transfected cells. The values were calculated as an average of 2 measurements from genomic DNA isolated from donors 4 to 6. The error bars represent the SD. (K) Frequency of indels, as measured by TIDE analysis, for ΔBCL11A (gRNA1617) of donors 4 to 6. (L) Percentage of F cells plotted as averages of 3 donors after 12 days of differentiation. A 1-way analysis of variance and Tukey’s post hoc test was used to calculated statistics. (M) HbF of donors 3 to 6 measured by HPLC 3 days after differentiation in control, ΔBCL11A, and Del2Rec conditions. We calculated the percentage of HbF over the total Hb tetramers. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. ns, nonsignificant; WT, wild-type.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal