Gates Package — page 845

Fig.5. Ex vivo HSC base editing for -globin reactivation by HDAd-EF1a.ABE8e. A) Schematic of the experiment. Bone marrow Lin- cells were isolated from -YAC/CD46transgenic mice and transduced ex vivo with HDAd-EF1a.ABE8e at an MOI of 500 vp/cell. After one day in culture, one million cells per mouse were transplanted into lethally irradiated C57Bl/6 mice, which were followed for 16 weeks ("week 16-P - primary"). Data from these mice are shown in this figure. Bone marrow Lin- cells from these mice were then used for secondary transplantation and these mice were monitored for another 16 weeks ("week 16-S-secondary", see Fig.S6). B) Engraftment of transplanted HDAdEF1a.ABE8e-transduced HSCs measured by flow cytometry of human CD46 in PBMCs. Each symbol is an individual mouse. N=5 animals. C) Analysis of target site editing in PBMCs by Sanger sequencing. Shown are percentages of conversion for the -113A>G site and neighboring adenines. N=5 animals. D) Analysis of target site editing in PBMCs, spleen, bone marrow MNCs, bone marrow lineage-negative cells and colony-forming units (CFU) at week 16 after transplantation by Sanger sequencing. N=5 animals. E) Comparison of editing rates (by next-generation sequencing - NGS) at the 4 adenines in the transplant (ex vivo transduced Lin- cells cultured for 3 days) and bone marrow MNCs at week 16 after transplantation. Shown are percentages of reads. Note the log10 scale of the Y-axis. N=3 animals. F) Next-generation sequencing of the target area (222 bp amplicon; ~100 nucleotides up- and downstream of the spacer). Left panel: Shown are base substitutions (green), deletions (blue), and insertions (red) in the target area for one representative mouse. Right panel: Summary of all indel reads in the transplant, week 16 primary mice and week 16 secondary mice. * p<0.05. Statistical analyses were performed using two-way ANOVA. G) Editing on a single cell basis. Week 16 bone marrow Lin- cells were plated for progenitor assay and individual colonies were subjected to NGS. Shown is a representative mouse with 100% bi-allelic editing of the HBG1/2 sites. n=36 (3 mice, 12 colonies per mouse analyzed).