Fig. 1

ZFN-mediated gene editing is highly efficient and leads to HbF induction independently of mobilization strategy or disease state. (a) Schematic of zinc-finger nucleases (ZFN) targeting the BCL11A erythroid-specific enhancer. The FokI domains dimerize over the core GATAA motif to induce double-strand breaks. Arrows represent the ZFN-nucleotide contacts. (b) Indel frequency and (c) cell viability using acridine orange/propidium iodide in single- (n = 5) and dual-mobilized (n = 4) CD34 + HSPC 2 days after ZFN editing of the BCL11A erythroid enhancer. (d) Gamma-globin (γ-globin) protein levels measured by reverse phase ultra-performance liquid chromatography (RP-UPLC) at day 21 of erythroid differentiation (single-mobilized n = 4, dual-mobilized n = 4). (e) Fraction of HbF⁺ cells in enucleated glycophorin A–positive erythroid cells derived from single- and dual-mobilized CD34 + HSPC (left; single-mobilized n = 3, dual-mobilized n = 2). A representative flow cytometry plot of HbF⁺ cell frequency is shown on the right. (f) Indel levels and (g) cell viability in HSPC derived from healthy (n = 4) and SCD (n = 3) donors 2 days post-transfection of ZFNs. (h) γ-globin protein levels measured by RP-UPLC at terminal erythroid differentiation (day 18). Data expressed as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001). G + P, granulocyte colony-stimulating factor plus plerixafor; HbF, fetal hemoglobin; HD, healthy donor; HSC, hematopoietic stem cell; P, plerixafor; RP-UPLC, reversed-phase ultra-performance liquid chromatography; SCD, sickle cell disease donor; SSC, side scatter; UT, untreated; ZFN, zinc finger nuclease.