Gates Package — page 824

was used. Results showed that no significant off-target base editing in the editable window (position 3 to 14) occurred at these 20 sites (Fig.9, middle panels). Notably, ~4% of alleles with single nucleotide variants in off-target site (OTS) #11 were found in both naïve and treated animals. Indel frequencies around the predicted nicking sites (70-bp at each side) of the 20 candidates showed no significant difference between naïve and treated animals (Fig.9, bottom panel). We conducted further off-target analyses using CasOFFinder, an in silico algorithm to predict potential off-target sites. A total of 76 and 89 sites with four or fewer mismatches to sgHBG#2 guide RNA were nominated in mouse and human genome, respectively (Figs.10A and B, Supplementary Excel file S2 and S3). Twenty four of the 76 Cas-OFFinder sites in the mouse genome overlapped with the CIRCLE-Seq OTS list (31.6%). The top 5 ranked sites from each list were amplified from the in vivo transduced mouse (analyzed above) or in vitro transduced thalassemia CD34+ cells for amplicon NGS. Results showed no significant editing at these sites. Effect on transcriptome. We compared RNA-Seq data of total RNA from bone marrow MNCs of CD46/- YAC mice before treatment and mice at week 16 after in vivo HSC transduction with HDAd-EF1.ABE8e (N=3 animals) (Fig.11, Suppl. Tables S5 and 6). Of the 2742 genes annotated to the human genome, only 13 were considered differentially expressed (adjusted p-value <0.01) (Suppl. Table S5). Six of them were upregulated and were clustered based on their biological pathways, into 3 groups: i) Hemoglobin genes, specifically HBG2, HBG1, and HBD. Their upregulation is most likely the result of in vivo editing of the - globin locus present in CD46/-YAC mice. ii) mgmt, as a result of residual expression of human mgmtP140K mRNA from the HDAd vector. Based on the mouse annotation process, we mapped 9037 genes in total, from which 7 were considered differentially expressed (adjusted p-value <0.01) (Suppl. Table S6). However, the level of upregulation was barely above the threshold set at 2.0-fold log. Of the seven genes, one gene is listed in the Catalogue of Somatic Mutations in Cancer (COSMIC). This gene, Creg1 (cellular repressor of E1A-stimulated genes), is upregulated in treated animals. Creg is considered to be a tumor suppressor and its upregulation should not be associated with cancer (39). Additional studies are required whether the modest upregulation of these genes is due to O6BG/BCNU treatment. Hematological analyses. There were no clinical side effects of the in vivo HSC transduction and selection approach. Histological analyses of major organs did not show abnormalities. To further assess the safety of in vivo base editing with HDAd-EF1.ABE8e, we performed hematological analyses of blood and bone marrow samples of week 16 in vivo transduced CD46/-YAC mice. No significant differences between