Figure 4.
Correction of the IVS1-110 (G>A) mutation in repopulating HSCs. (A) Overview of the experimental protocol of HSPC xenotransplantation. BT HSPCs were subjected to RNA-mediated base editing and xenotransplanted into NBSGW immunodeficient mice. HD and BT HSPCs electroporated with TE buffer or only with SpRY-ABE8e mRNA were injected as controls. Peripheral blood analysis was performed at weeks 9 and 16. Mice were euthanized 16 weeks after engraftment, after Clo-Lip injection, and their hematopoietic tissues and organs were collected and analyzed. (B) Engraftment of human cells in NBSGW mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks post-transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). Engraftment is represented as the percentage of human CD45+ cells in the total murine and human CD45+ cell population in peripheral blood, bone marrow, spleen, and thymus. Each data point represents an individual mouse. The mouse with the lowest chimerism is indicated with the symbol ◓. Data are expressed as mean ± SEM. (C) Frequency of human T (CD3) and B (CD19) lymphoid, myeloid (CD14, CD15, and CD11b), erythroid (GPA, CD36, CD71), and HSPC (CD34) cells in BM 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). Each data point represents an individual mouse. Data are expressed as mean ± SEM. (D) Human hematopoietic progenitor content in BM human CD45+ cells derived from mice transplanted with control and edited HSPCs (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). We plotted the percentage of human CD45+ cells giving rise to BFU-E and CFU-GM. Data are expressed as mean ± SEM. (E) Base editing efficiency, calculated by the EditR software, in input, peripheral blood-, bone marrow- and spleen-derived HD, and BT human samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (BT-cor, n = 5). The frequency of base editing in the input was calculated in cells cultured in the HSPC medium (▲), in liquid erythroid cultures (▼), BFU-E (■) and CFU-GM (◆) colonies. Each data point represents an individual mouse. Data are expressed as mean ± SEM. ctr, control. BFU-E, burst forming units-erythroid; CFU-GM, colony forming units-erythroid.

Correction of the IVS1-110 (G>A) mutation in repopulating HSCs. (A) Overview of the experimental protocol of HSPC xenotransplantation. BT HSPCs were subjected to RNA-mediated base editing and xenotransplanted into NBSGW immunodeficient mice. HD and BT HSPCs electroporated with TE buffer or only with SpRY-ABE8e mRNA were injected as controls. Peripheral blood analysis was performed at weeks 9 and 16. Mice were euthanized 16 weeks after engraftment, after Clo-Lip injection, and their hematopoietic tissues and organs were collected and analyzed. (B) Engraftment of human cells in NBSGW mice transplanted with HD or BT control (HD-ctr; BT-ctr) or corrected (BT-cor) HSPCs 16 weeks post-transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). Engraftment is represented as the percentage of human CD45+ cells in the total murine and human CD45+ cell population in peripheral blood, bone marrow, spleen, and thymus. Each data point represents an individual mouse. The mouse with the lowest chimerism is indicated with the symbol ◓. Data are expressed as mean ± SEM. (C) Frequency of human T (CD3) and B (CD19) lymphoid, myeloid (CD14, CD15, and CD11b), erythroid (GPA, CD36, CD71), and HSPC (CD34) cells in BM 16 weeks after the transplantation (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). Each data point represents an individual mouse. Data are expressed as mean ± SEM. (D) Human hematopoietic progenitor content in BM human CD45+ cells derived from mice transplanted with control and edited HSPCs (HD-ctr, n = 4; BT-ctr, n = 4; BT-cor, n = 5). We plotted the percentage of human CD45+ cells giving rise to BFU-E and CFU-GM. Data are expressed as mean ± SEM. (E) Base editing efficiency, calculated by the EditR software, in input, peripheral blood-, bone marrow- and spleen-derived HD, and BT human samples subjected to Sanger sequencing. Data are expressed as mean ± SEM (BT-cor, n = 5). The frequency of base editing in the input was calculated in cells cultured in the HSPC medium (▲), in liquid erythroid cultures (▼), BFU-E (■) and CFU-GM (◆) colonies. Each data point represents an individual mouse. Data are expressed as mean ± SEM. ctr, control. BFU-E, burst forming units-erythroid; CFU-GM, colony forming units-erythroid.

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