Functional correction of sickle bone marrow CD34⁺ cells. Bone marrow CD34⁺ cells from patients with SCD were electroporated with in vitro-transcribed ZFN mRNA and transduced with donor IDLV carrying the WT base at the sickle location and grown under erythroid conditions. (A) Correction at the sickle mutation evaluated by high-throughput sequencing. Results of sequencing of the β-globin locus showing percentage of total aligned reads containing the corrected WT base (A) at the sickle mutation, as well as insertions and deletions (indels) at the cut site. Corrected base, white; indels, gray. (B) HPLC of differentiated erythroid cells at the termination of culture. Cells were pelleted and lysed, and supernatant was analyzed by HPLC. (Left) SCD mock sample. (Right) SCD ZFN+IDLV sample. Shading indicates HbA:WT adult hemoglobin peak. (C) Quantification of the percent of HbA out of the total area under the curve represented by the main peaks. HbA, WT adult hemoglobin; HbF, fetal hemoglobin; HbS, sickle hemoglobin; n.s., not significant (n = 2 independent experiments; mock, n = 3; ZFN only, n = 4; IDLV only, n = 3; ZFN+IDLV, n = 6).