American Society of Hematology

Figure 3.

$Xenotransplantation of normal gene-edited CD34+cells into NBSGW mice. Peripheral blood (PB) CD34+ cells were edited with Cas9:sgRNA-1 RNP using the Neon Transfection System and then transplanted into NBSGW mice via tail-vein injection. Donor cell controls (C) were processed in parallel, but not electroporated. Donor-cell progeny were analyzed in recipient peripheral blood at 6, 11, and 16 weeks and in bone marrow (BM) at 17 weeks after transplantation. Data are from 3 independent experiments with CD34+ cells from different donors (red, blue, or green). Each dot in graphs represents a separate mouse. (A) Normalized human chimerism in peripheral blood and bone marrow, shown as the percentage of human (h) CD45+ cells. (B) Human T (CD3+), B (CD19+), and myeloid (M) (CD33+) cells shown as percentages of the human CD45+ population in bone marrow at 16 weeks. Erythroid chimerism (E) is shown as the percentage of human CD235a+ cells within the CD45− population (mouse and human). (C) The indel fraction 4 days after editing (Pre BMT) and as measured serially after xenotransplantation. Dark blue represents the 13-nt human HPFH mutation; light blue represents all other indels. (D) Indels in specific hematopoietic lineages. Dark blue represents the 13-nt human HPFH mutation; light blue represents all other indels. (E) The most frequent indels in donor 1 CD34+ cells before bone marrow transplantation and in the 16-week bone marrow of mouse recipients. Unique indels are shown with a different color, with the bar size representing the relative abundance. The 13-nt HPFH mutation is represented in black. All indels present in proportions <1% are represented collectively in blue at the top of each bar. (F) Morphology of human CD235a+ erythroblasts in bone marrow of recipient mice transplanted with RNP-edited or control CD34+ cells. The images were acquired with a Nikon Eclipse Ni microscope with a 60× objective and Nikon NIS-Elements software (scale bars, 10 μm). (G) Flow cytometry for the erythroid maturation markers CD49d and Band3 on human CD235a+ erythroblasts in recipient mouse bone marrow. (H) Enucleated (HOECHST−) and nucleated (HOECHST+) human CD235a+ erythroblasts in recipient bone marrow. The numbers in panels G-H represent the mean ± SD of the percentage for the same samples analyzed in panels A-D. (I) The fraction of CD235a+ F-cells in bone marrow. (J) The %HbF in human CD235a+ erythroblasts isolated from recipient bone marrow. (K) The %HbF as a function of the indel percentage from the values plotted in panels C and J. The bar charts in panels A-D and I-J show the mean ± SD percentages for 3 biological replicates performed using CD34+ cells from 3 donors. Each dot represents a single recipient mouse, and each color represents a separate replicate study. ****P < .0001 (by unpaired Student t test).$

Xenotransplantation of normal gene-edited CD34⁺cells into NBSGW mice. Peripheral blood (PB) CD34⁺ cells were edited with Cas9:sgRNA-1 RNP using the Neon Transfection System and then transplanted into NBSGW mice via tail-vein injection. Donor cell controls (C) were processed in parallel, but not electroporated. Donor-cell progeny were analyzed in recipient peripheral blood at 6, 11, and 16 weeks and in bone marrow (BM) at 17 weeks after transplantation. Data are from 3 independent experiments with CD34⁺ cells from different donors (red, blue, or green). Each dot in graphs represents a separate mouse. (A) Normalized human chimerism in peripheral blood and bone marrow, shown as the percentage of human (h) CD45⁺ cells. (B) Human T (CD3⁺), B (CD19⁺), and myeloid (M) (CD33⁺) cells shown as percentages of the human CD45⁺ population in bone marrow at 16 weeks. Erythroid chimerism (E) is shown as the percentage of human CD235a⁺ cells within the CD45⁻ population (mouse and human). (C) The indel fraction 4 days after editing (Pre BMT) and as measured serially after xenotransplantation. Dark blue represents the 13-nt human HPFH mutation; light blue represents all other indels. (D) Indels in specific hematopoietic lineages. Dark blue represents the 13-nt human HPFH mutation; light blue represents all other indels. (E) The most frequent indels in donor 1 CD34⁺ cells before bone marrow transplantation and in the 16-week bone marrow of mouse recipients. Unique indels are shown with a different color, with the bar size representing the relative abundance. The 13-nt HPFH mutation is represented in black. All indels present in proportions <1% are represented collectively in blue at the top of each bar. (F) Morphology of human CD235a⁺ erythroblasts in bone marrow of recipient mice transplanted with RNP-edited or control CD34⁺ cells. The images were acquired with a Nikon Eclipse Ni microscope with a 60× objective and Nikon NIS-Elements software (scale bars, 10 μm). (G) Flow cytometry for the erythroid maturation markers CD49d and Band3 on human CD235a⁺ erythroblasts in recipient mouse bone marrow. (H) Enucleated (HOECHST⁻) and nucleated (HOECHST⁺) human CD235a⁺ erythroblasts in recipient bone marrow. The numbers in panels G-H represent the mean ± SD of the percentage for the same samples analyzed in panels A-D. (I) The fraction of CD235a⁺ F-cells in bone marrow. (J) The %HbF in human CD235a⁺ erythroblasts isolated from recipient bone marrow. (K) The %HbF as a function of the indel percentage from the values plotted in panels C and J. The bar charts in panels A-D and I-J show the mean ± SD percentages for 3 biological replicates performed using CD34⁺ cells from 3 donors. Each dot represents a single recipient mouse, and each color represents a separate replicate study. ****P < .0001 (by unpaired Student t test).

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