American Society of Hematology

Figure 6.

$HBG1 and HBG2 promoters in CD34+HSPCs from individuals with SCD induces HbF expression in erythroid progeny generated after xenotransplantation. Plerixafor-mobilized CD34+ cells from 2 individuals with SCD (represented by blue and red) were edited with Cas9:sgRNA-1 RNP by using the Neon Transfection System. They were then transplanted into NBSGW mice via tail-vein injection. Donor-cell controls (C) were processed in parallel, but not electroporated. Donor cells were analyzed in recipient bone marrow at 16 to 18 weeks after transplantation. (A) Normalized human cell chimerism (hCD45%) in bone marrow for the 2 SCD donors. (B) Chimerism for specific human lineages in recipient bone marrow, labeled as in Figure 3B. (C) The indel fraction in CD34+ cells at 4 days after gene editing (Pre) and in bone marrow after xenotransplantation. Dark blue represents the fraction of indels with the 13-nt HPFH deletion; light blue represents all other indels. (D) Large deletions were quantified by the ΔHBG2 and Δ366+ assays (Figure 4A) in edited CD34+ cells before 16 weeks after transplantation. (E) Morphology of human CD235a+ erythroblasts in bone marrow of mice transplanted with control or RNP-edited CD34+ cells. The images were acquired using a Nikon Eclipse Ni microscope with a 60× objective and Nikon NIS-Elements software (scale bars, 10 μm). (F) The F-cell fraction in bone marrow CD235a+ erythroblasts. (G) The %HbF in CD235a+ erythroblasts isolated from recipient bone marrow, as determined by ion-exchange HPLC. (H) The correlation of the %indels with the %HbF in CD235a+ erythroblasts isolated from recipient bone marrow. (I) Human erythroblasts isolated from mouse recipient bone marrow were incubated for 8 hours in 2% O2 and visualized by phase-contrast microscopy using the IncuCyte S3 Live-Cell Analysis System (Sartorius) with a 20× objective. The white arrowheads indicate cells with sickle-like morphology. (J) The sickled cell fraction after hypoxia in edited and control cells was determined by analyzing micrographs such as those in panel I. More than 300 cells in 2 independent experiments were scored by 2 blinded observers. All bar charts show data as the mean ± SD. Each dot represents a single recipient mouse from donor 1 (blue) or 2 (red). ****P < .0001, ***P < .001, **P < .01, and *P < .05 (by unpaired Student t test).$

HBG1 and HBG2 promoters in CD34⁺HSPCs from individuals with SCD induces HbF expression in erythroid progeny generated after xenotransplantation. Plerixafor-mobilized CD34⁺ cells from 2 individuals with SCD (represented by blue and red) were edited with Cas9:sgRNA-1 RNP by using the Neon Transfection System. They were then transplanted into NBSGW mice via tail-vein injection. Donor-cell controls (C) were processed in parallel, but not electroporated. Donor cells were analyzed in recipient bone marrow at 16 to 18 weeks after transplantation. (A) Normalized human cell chimerism (hCD45%) in bone marrow for the 2 SCD donors. (B) Chimerism for specific human lineages in recipient bone marrow, labeled as in Figure 3B. (C) The indel fraction in CD34⁺ cells at 4 days after gene editing (Pre) and in bone marrow after xenotransplantation. Dark blue represents the fraction of indels with the 13-nt HPFH deletion; light blue represents all other indels. (D) Large deletions were quantified by the ΔHBG2 and Δ366⁺ assays (Figure 4A) in edited CD34⁺ cells before 16 weeks after transplantation. (E) Morphology of human CD235a⁺ erythroblasts in bone marrow of mice transplanted with control or RNP-edited CD34⁺ cells. The images were acquired using a Nikon Eclipse Ni microscope with a 60× objective and Nikon NIS-Elements software (scale bars, 10 μm). (F) The F-cell fraction in bone marrow CD235a⁺ erythroblasts. (G) The %HbF in CD235a⁺ erythroblasts isolated from recipient bone marrow, as determined by ion-exchange HPLC. (H) The correlation of the %indels with the %HbF in CD235a⁺ erythroblasts isolated from recipient bone marrow. (I) Human erythroblasts isolated from mouse recipient bone marrow were incubated for 8 hours in 2% O₂ and visualized by phase-contrast microscopy using the IncuCyte S3 Live-Cell Analysis System (Sartorius) with a 20× objective. The white arrowheads indicate cells with sickle-like morphology. (J) The sickled cell fraction after hypoxia in edited and control cells was determined by analyzing micrographs such as those in panel I. More than 300 cells in 2 independent experiments were scored by 2 blinded observers. All bar charts show data as the mean ± SD. Each dot represents a single recipient mouse from donor 1 (blue) or 2 (red). ****P < .0001, ***P < .001, **P < .01, and *P < .05 (by unpaired Student t test).

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