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HbF rescues dyserythropoiesis in SCD
CD
repressor LRF in the fetal γ-globin promoters (HBG1 and HBG2)12 (Online Supplementary Figure S4C). CD34+ cells were transfected either with a gRNA targeting the LRF binding site (-197) or a gRNA targeting an unrelated locus (AAVS1). Genome editing efficiency at the γ-globin pro- moter ranged between 23.2% and 73.5%, as determined by Sanger sequencing at D6 of phase I of culture. Cells were grown and differentiated under normoxia or hypox- ia. As expected, the disruption of the LRF binding site resulted in HbF induction as shown by higher %F-cells compared to AAVS1 control (Figure 6C; Online Supplementary Figure S4D). These higher levels of F-cells resulted in decreased apoptosis, under both normoxic and hypoxic conditions (Figure 6D; Online Supplementary Figure S4E), clearly demonstrating the positive and selective effect of HbF on SCD cell survival.
Discussion
Ineffective erythropoiesis has been previously suggest- ed to be a feature of SCD7-9,27 but it has not been critically evaluated and documented. Our present findings provide direct evidence for ineffective erythropoiesis in SCD patients, with significant cell death occurring at the late
Figure 2. (continued from previous page). (D) Percentage of dead cells (means ± standard error of the mean) measured in the F- and F+ subpopulations for control and patient cells under hypoxia. *P<0.05, **P<0.01, ****P<0.0001; Wilcoxon paired test and Mann-Whitney test (C); Mann-Whitney test (D)
stages of terminal erythroid differentiation in vivo. Among previous reports, the study by Wu and collab- orators has highlighted abnormalities during erythro- poiesis in the bone marrow of transplanted SCD patients by showing progressive intramedullary loss of SCD ery- throblasts and relative enrichment of donor erythroid precursors at the onset of hemoglobinization in a small cohort of patients.9 Our results further demonstrate sig- nificant cell death between the polychromatic and the orthochromatic stages, when cellular HbS concentration reaches sufficiently high levels to promote polymer for- mation under partial hypoxia. Our in vivo data unequivo- cally document the occurrence of ineffective erythro- poiesis in non-transplanted SCD patients, in the absence of confounding factors like conditioning drugs and exogenous donor-related factors that can impact the hematopoietic niche and interfere with the survival of
patient’s erythroblasts.
This study also reveals a new role for HbF in SCD by
showing that it protects a subpopulation of differentiat- ing erythroblasts from apoptosis, both in vivo and in vitro. HbF is a known modulator of disease severity in SCD as it inhibits HbS polymerization at the molecular level, preventing or attenuating RBC sickling and alleviating disease complications.28,29 In healthy adults, HbF accounts
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