Transplantation of ZFN and donor-treated cells into NSG mice. (A) Gene modification rates of bulk transplanted cells treated with ZFN+IDLV and cultured in vitro as determined by qPCR for the RFLP at 7 days after electroporation. Mock cells are untreated (n = 3 independent experiments). (B) Modification at the sickle base evaluated by high-throughput sequencing for ZFN+IDLV-modified CD34⁺ cells. Results of sequencing of the β-globin locus showing percentage of total aligned reads containing the changed wild-type to sickle base (T), as well as insertions and deletions (indels) at the cut site. Same samples as in A. Changed base, white; indels, gray. (C) CD34⁺ cells were electroporated with Oligo, ZFN, or ZFN+Oligo and cultured in vitro before transplantation into NSG mice. Modification rates at the sickle base and indels are shows as in B (n = 1 experiment). (D) Engraftment in the peripheral blood of transplanted mice at 5 and 8 weeks after transplant. Human engraftment determined as a percentage of hCD45⁺ cells out of the total hCD45⁺ and mCD45⁺ cells by flow cytometry of cells from mice receiving either mock- or ZFN+IDLV-treated cells. Mock, open diamonds; ZFN+IDLV, closed diamonds. (n = 3 independent experiments; mock, n = 5; ZFN+IDLV, n = 12; unpaired t test). (E) Engraftment in the peripheral blood as in D of cells from mice receiving either Oligo-, ZFN-, or ZFN+Oligo-treated cells. Oligo, circles; ZFN, triangles; ZFN+Oligo, diamonds (Oligo, n = 8; ZFN, n = 7; ZFN+Oligo, n = 9); 1-way analysis of variance. n.s., not significant. *P < .05, **P < .01. Error bars, mean ± standard deviation.