after the second round of selection and reached levels above 80% after the third round. The percentage of γ-globin-expressing cells was approximately 7-to 10-fold higher in erythroid Ter119 + cells versus nonerythroid Ter119cells in peripheral blood and bone marrow (Figure 1D). We used HPLC to measure the level of γ-globin protein in comparison with the adult mouse α-and β-globin chains (Figure 1E and Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/ JCI122836DS1). At week 18, these levels reached 10%-15% of adult mouse α-globin and β-major globin and approximately 25% of mouse β-minor globin. This was confirmed on the mRNA level by quantitative reverse transcription PCR (RT-qPCR), where human γ-globin mRNA was approximately 13% of mouse β-major mRNA (Figure 1F). To further demonstrate that primitive, long-term repopulating HSCs were transduced, we transplanted lineagedepleted (Lin-) bone marrow cells from in vivo-transduced/ selected mice into irradiated C57BL/6 mice. Engraftment levels analyzed in peripheral blood, bone marrow, and spleen were greater than 95% and stable over an observation period of 20 weeks (Supplemental Figure 2, A and B). Human γ-globin levels (compared with mouse α-globin) were similar in ("primary") in vivo-transduced mice (analyzed at week 18 after transduction) and secondary recipients analyzed at weeks 14 and 20 after transplantation (Supplemental Figure 2C). The in vivo HSPC transduction/selection approach does not change the SB100X-mediated random transgene integration pattern and does not alter hematopoiesis. We previously showed that in vivo transduction with the hybrid transposon/SB100X HDAd5/35++ system resulted in random transgene integration in HSPCs (6). To evaluate the effect of O 6 BG/BCNU in in vivo selection, we analyzed transgene integration in bone marrow Lincells at the end of the study, i.e., at week 20 in secondary recipients. Linear amplification-mediated PCR (LAM-PCR) followed by deep sequencing showed a random distribution pattern of integration sites in the mouse genome (Figure 2A). Data pooled from 5 mice demonstrated 2.23% integration into exons, 31.58% into introns, 65.17% into intergenic regions, and 1.04% into untranslated regions (Figure 2B). The level of randomness of integration was 99% without preferential integration in any given window of the whole mouse genome (Figure 2C). This indicates that in vivo selection and further expansion of cells in secondary recipients did not result in the emergence of dominant integration sites (Figure 2D). We measured, by qPCR, on average two γ-globin cDNA copies per bone marrow cell in a population containing both transduced and nontransduced cells. We then quantified the integrated transgene copy number on a single-cell level. To do this, we plated bone marrow Lincells from week 18 mice in methylcellulose, isolated individual progenitor colonies, and performed qPCR on genomic DNA. In transgene-positive colonies (n = 113), 86.7% of colonies had 2 or 3 integrated co...

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