Fig. 3

ZFN-mediated gene editing does not compromise the functionality or progenies of HSPC and results in stable editing levels after engraftment in vivo. (a) PB indel levels at input (Week 0, n = 2 donors) and after engraftment (For each donor, n = 9–10 mice for week 8 and 12 post engraftment, and n = 4–5 mice for week 16 and 19). (b) Indel levels in human bone marrow (BM) as well as cell subpopulations within the hCD45 + compartment sorted using antibodies recognizing the indicated lineage markers at input (Week 0) and after engraftment (Week 19 post engraftment). No significant difference was observed between lineages at week 19 using a multivariate ANOVA controlling for donor differences (P > 0.05, excluding input). BM refers to the total human CD45 + cell population in the bone marrow. (c) Frequency of enucleated cells in terminal erythroid differentiation in vitro from BM-derived HSPC at Week 19. (d) Globin protein levels were analyzed at Day 16 of in vitro erythroid differentiation by UPLC. Average γ-globin protein levels for each group are plotted as percentage of all β-like globin protein (A-γ-, G-γ-, δ-, and adult β-globin protein) on the left, and average γ-globin protein levels as percentage of α-globin levels are plotted on the right. (e) Read counts of the 10 most frequent sequences identified by next-generation sequencing at input (Week 0) in healthy donor 1 (HD 1) and healthy donor 2 (HD 2). The unedited (wild type) sequence is shown in bold, with a rectangle highlighting the core GATAA site and lines representing ZFN contacts. (f) Fraction of total indels that are likely derived from MMEJ. MMEJ patterns represent those identified by the RGEN Microhomology-Predictor tool (see Methods). (g) Frequency of indels by size of insertion (> 0) or deletions (< 0). Data expressed as mean ± standard deviation (*P < 0.05; **P < 0.01; ***P < 0.001). HD, healthy donor; HSC, hematopoietic stem cell; MMEJ, microhomology-mediated end-joining; n.s., not significant; PB, peripheral blood; BM, bone marrow; UPLC; ultra-performance liquid chromatography; UT, untreated; ZFN, zinc finger nuclease.