Extended Data Figure 6. Analysis of human engraftment.

(A) Representative FACS plot from the analysis of the bone marrow of a control mouse not transplanted with human cells. Mice were sacrificed and bone marrow was harvested from femur, tibia, hips, humerus, sternum, and vertebrae. Cells were subject to Ficoll density gradient to isolate mononuclear cells, which were analyzed for human engraftment by flow cytometry. Human engraftment was delineated as huCD45/HLA-ABC double positive. 4 out of 157,898 cells were found within the human cell gate. (B) Representative FACS plots showing gating scheme for analyses of NSG mice transplanted with human cells and analyzed as described in (A). Representative plots are from one mouse from the RNP + rAAV6 experimental group. As above, human engraftment was delineated as huCD45/HLA-ABC double positive. B cells were marked by CD19 expression, and myeloid cells identified by CD33 expression. GFP expression was analyzed in total human cells (2.4%), B-cells (1.9%) and myeloid cells (2.8%). The GFP brightness in B cells is lower than in myeloid cells suggesting that the SFFV promoter is not as active in the B-cell lineage compared to the myeloid lineage (see also Fig 4a). (C) Overview of engraftment for RNP+AAV and RNP+AAV GFP^high experimental groups. Average engraftment frequencies and percent GFP⁺ human cells +/-S.E.M are shown. Total number of cells transplanted was the same (500,000) for all mice in the RNP group, whereas in the GFP^high group, one mouse was transplanted with 100,000 cells, two mice with 250,000 cells, and three mice with 500,000 cells. The total number of HSCs transplanted per mouse (+/-S.E.M.) was calculated based on the frequencies of GFP⁺ cells in the CD34⁺/CD38^-/CD90⁺/CD45RA^- subset analyzed by flow cytometry (see Fig 3d) directly before injection. The total number of modified human cells in the bone marrow (BM) at Week 16 post transplant per mouse (+/-S.E.M.) was estimated based on calculations presented in the materials and methods. This shows that the enrichment not only resulted in a higher percentage of edited cells (column 3) but also resulted in an absolute higher number (column 6) of edited cells as well.