Figure 5.
Correction of the IVS1-110 (G>A) mutation in xenotransplanted BT HSPCs rescues the ineffective erythropoiesis in vivo. (A) Frequency of ROS-containing (DCFDA+) human GPA+ erythroid cells derived from the bone marrow of mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 3; BT-ctr, n = 4; BT-cor, n = 4). ∗∗P ≤ .01 (unpaired t test; BT-ctr vs BT-cor). We plotted the fold change relative to BT-ctr samples. (B) Frequency of enucleated cells as measured by the flow cytometry analysis of cells stained with the DRAQ5 nuclear dye in human GPA+ erythroid populations from the bone marrow of mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 3; BT-ctr, n = 4; BT-cor, n = 4). ∗P ≤ .05 (unpaired t test; BT-ctr vs BT-cor). (C) Representative RP-HPLC chromatograms from sorted human GPA+ bone marrow erythroid cells 16 weeks posttransplantation. (D) α/non–α ratio calculated based on RP-HPLC data from sorted human GPA+ bone marrow erythroid cells obtained from mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n=5). The dotted lines indicate minimum and maximum values observed in HD-ctr samples. ∗P ≤ .05 (unpaired t test; BT-ctr vs BT-cor). (E) Frequency of human RBCs in total peripheral blood 4 days after Clo-Lip injection in mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). (F) Representative RP-HPLC chromatograms from sorted human circulating RBCs 16 weeks after the transplantation (BT-ctr, n = 4 pooled samples; BT-cor, n = 1 representative graph; HD-ctr, n = 1 representative graph). cor, corrected; ctr, control.

Correction of the IVS1-110 (G>A) mutation in xenotransplanted BT HSPCs rescues the ineffective erythropoiesis in vivo. (A) Frequency of ROS-containing (DCFDA+) human GPA+ erythroid cells derived from the bone marrow of mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 3; BT-ctr, n = 4; BT-cor, n = 4). ∗∗P ≤ .01 (unpaired t test; BT-ctr vs BT-cor). We plotted the fold change relative to BT-ctr samples. (B) Frequency of enucleated cells as measured by the flow cytometry analysis of cells stained with the DRAQ5 nuclear dye in human GPA+ erythroid populations from the bone marrow of mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 3; BT-ctr, n = 4; BT-cor, n = 4). ∗P ≤ .05 (unpaired t test; BT-ctr vs BT-cor). (C) Representative RP-HPLC chromatograms from sorted human GPA+ bone marrow erythroid cells 16 weeks posttransplantation. (D) α/non–α ratio calculated based on RP-HPLC data from sorted human GPA+ bone marrow erythroid cells obtained from mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n=5). The dotted lines indicate minimum and maximum values observed in HD-ctr samples. ∗P ≤ .05 (unpaired t test; BT-ctr vs BT-cor). (E) Frequency of human RBCs in total peripheral blood 4 days after Clo-Lip injection in mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). (F) Representative RP-HPLC chromatograms from sorted human circulating RBCs 16 weeks after the transplantation (BT-ctr, n = 4 pooled samples; BT-cor, n = 1 representative graph; HD-ctr, n = 1 representative graph). cor, corrected; ctr, control.

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