Figure 1.
Characterization of WT, Hba-a1Fl/Fl/Hba-a2KO/KO + mRNACre-LNPCD117 (AG-KO), and HSC Hba-a1Fl/Fl/Hba-a2KO/KO + mRNACre-LNPCD117 + ALS20αI (AG-KOALS20αI) transplant recipients. (A) Scheme showing the Cre-mediated excision of Hba-a1 after administration of mRNACre-LNPCD117. Although administration of mRNACreLNPCD117 offers impressive in vivo recombination, we opted for in vitro treatment and engraftment of isolated lin− cells to ensure the near total gene deletion. (B) Agarose gels showing the deletion of the Hba-a1 (αF) genes in cells collected and kept in vitro after lin− cell collection. Amplification after recombination results in a 496-base pair (bp) product, whereas the absence of recombination (no cells exposed to LNP) does not produce an amplification because of size constraints. The deletion was observed after treatment with mRNACre-LNPCD117 and keeping the cells in culture, as indicated. Only relevant lanes are shown; the full gel is shown in supplemental Figure 37. The black line separates 2 different sections of the gel. (C) Levels of endogenous genomic mouse Hba (Hba-a1 + Hba-a2), after treatment with mRNACre-LNPCD117 measured by droplet digital polymerase chain reaction (ddPCR). The ddPCR assay amplifies any remaining intact genomic α-globin gene, either Hba-a1 or Hba-a2. The residual genomic α-globin amplification in AG-KO level mice is compared with nonrecombined WT control (4 copies of the α-globin genes) and mice carrying 3 or 2 copies of the α-globin genes, shown as a percentage. The WT is set to 100% by default. (D) Cation-exchange HPLC chromatograms of WT, AG-KO, and AG-KOALS20αI mice that received transplantation were analyzed. Absolute counts of erythroblasts in the BM (E) and the spleen (F) include proerythroblasts (I, in purple), basophilic erythroblasts (II, in blue), polychromatic erythroblasts (III, in yellow), orthochromatic erythroblasts and reticulocytes (IV, in green), and RBC (V, in red). Significance was assessed by ordinary 2-way analysis of variance (ANOVA) using Tukey multiple comparisons test (n = 4-6; test described in supplemental Table 1). (G) Oxygen equilibrium curves of WT and AG-KO mice, as well as AG-KOALS20αI with VCN in the range of 1.1 to 2.9, values were extrapolated using the method described in supplementary Materials, in “Oxygen binding affinity.”

Characterization of WT, Hba-a1Fl/Fl/Hba-a2KO/KO + mRNACre-LNPCD117 (AG-KO), and HSC Hba-a1Fl/Fl/Hba-a2KO/KO + mRNACre-LNPCD117 + ALS20αI (AG-KOALS20αI) transplant recipients. (A) Scheme showing the Cre-mediated excision of Hba-a1 after administration of mRNACre-LNPCD117. Although administration of mRNACreLNPCD117 offers impressive in vivo recombination, we opted for in vitro treatment and engraftment of isolated lin− cells to ensure the near total gene deletion. (B) Agarose gels showing the deletion of the Hba-a1 (αF) genes in cells collected and kept in vitro after lin− cell collection. Amplification after recombination results in a 496-base pair (bp) product, whereas the absence of recombination (no cells exposed to LNP) does not produce an amplification because of size constraints. The deletion was observed after treatment with mRNACre-LNPCD117 and keeping the cells in culture, as indicated. Only relevant lanes are shown; the full gel is shown in supplemental Figure 37. The black line separates 2 different sections of the gel. (C) Levels of endogenous genomic mouse Hba (Hba-a1 + Hba-a2), after treatment with mRNACre-LNPCD117 measured by droplet digital polymerase chain reaction (ddPCR). The ddPCR assay amplifies any remaining intact genomic α-globin gene, either Hba-a1 or Hba-a2. The residual genomic α-globin amplification in AG-KO level mice is compared with nonrecombined WT control (4 copies of the α-globin genes) and mice carrying 3 or 2 copies of the α-globin genes, shown as a percentage. The WT is set to 100% by default. (D) Cation-exchange HPLC chromatograms of WT, AG-KO, and AG-KOALS20αI mice that received transplantation were analyzed. Absolute counts of erythroblasts in the BM (E) and the spleen (F) include proerythroblasts (I, in purple), basophilic erythroblasts (II, in blue), polychromatic erythroblasts (III, in yellow), orthochromatic erythroblasts and reticulocytes (IV, in green), and RBC (V, in red). Significance was assessed by ordinary 2-way analysis of variance (ANOVA) using Tukey multiple comparisons test (n = 4-6; test described in supplemental Table 1). (G) Oxygen equilibrium curves of WT and AG-KO mice, as well as AG-KOALS20αI with VCN in the range of 1.1 to 2.9, values were extrapolated using the method described in supplementary Materials, in “Oxygen binding affinity.”

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