Gates Package — page 849

Fig.7. In vivo HSC transduction of -YAC/CD46 mice to achieve -globin reactivation by HDAd-EF1a.ABE8e. A) Experimental procedure. β-YAC/CD46 mice (N=5 animals) were mobilized by G-CSF/AMD3100 and in vivo transduced by intravenous (IV) injection of HDAd-EF1a.ABE8e. In vivo selection with O6BG/BCNU was started at day 2 after HDAd injection and repeated at days 12 and 26 at the indicated doses (30mg/kg O6BG and 5, 10, 10mg/kg BCNU). The mice were euthanized at week 16 ("week 16-P"). The data from "primary" in vivo transduced mice are sown in this figure. Lineage-negative cells were isolated from bone marrow and IV injected into lethally irradiated C57BL/6J mice. The secondary transplanted mice were followed for another 16 weeks ("week 16-S", see Fig.S10). B) Loss of vector genomes in PBMCs and bone marrow MNCs. Vector copy number (VCN) was measured by qPCR with human mgmtP140K primers. ND: not detectable. N=5 animals for d3 and wk16; N=3 animals for wk4 and wk6. C-E) Target base conversion measured by Sanger sequencing. Each dot represents one animal. N=5 animals. C) Percent conversion in DNA from PBMCs. D) Percent conversion at week 16 in PBMCs and MNCs in the spleen and bone marrow. E) Percent conversion at week 16 in lineage (CD3+, CD19+, Gr-1+, Ter119+) cells and in Lin- cells in the bone marrow. F) Comparison of editing rates at the 4 adenines in Bone marrow MNCs at week 16 after in vivo transduction. Shown are percentages of reads. N=5 animals. G) Representative NSG results showing target base conversion and indels at week 16 (mouse #446). H) Summary of all indel reads in week 16 primary mice and week 16 secondary mice. N=5 animals. I) Editing on a single cell basis. Week 16 bone marrow Lin- cells were plated for progenitor assays and individual colonies were subjected to NGS. Shown are sequencing data of the HBG1 and HBG2 sites of a representative colony with mono- and bi-allelic conversions as well as indels (<1%, not visible).