Therapeutic prime editing in CD46/Townes mice after in vivo HSC transduction with HDAd-PE5max. (A) Experimental procedure. Mice (n = 7) were mobilized using G-CSF/AMD3100 and were transduced in vivo with HDAd-PE5max (8 × 10¹⁰ vp/mouse = 3.2 × 10¹² vp/kg). For in vivo selection, mice were injected with O⁶-BG (15 mg/kg, intraperitoneal [IP]) 2 times, 30 minutes apart. One hour after the second injection of O⁶-BG, mice were injected with BCNU (5 mg/kg, IP). For the second and third cycle, BCNU concentrations were increased to 9 mg/kg and 10 mg/kg, respectively. The mice were killed during week 16 for analyses. Lin^– cells were isolated from BM and IV injected into lethally irradiated C57BL/6J mice. The secondary transplanted mice were followed for another 16 weeks for the specified terminal point analyses. (B-F) Editing measured using Sanger sequencing of in vivo–transduced mice. (B) Target base editing (sickle repair and silent PAM) in PBMCs. (C) Editing in cells from different tissues at week 16 after in vivo transduction. (D) Editing in different lineage-positive cell subsets and Lin^– cells at week 16. CD3⁺ T cells, CD19⁺ B cells, Gr1⁺ granulocytes, Ter-119⁺ erythroid cells were sorted from BM MCs using flow cytometry. (E) Allelic analysis in single Lin^– cell–derived progenitor colonies. Editing was measured at day 11 after plating of transduced Lin^– cells. Data from 4 mice with indicated ear tag number are shown. Twelve colonies for each mouse were analyzed. (F) Editing in tissues (n = 4). (G-I) Analyses of secondary recipients. Lin^– cells from in vivo–transduced mice were transplanted into lethally irradiated C57BL/6 mice (n = 7) (cells from donor into 1 recipient). (G) Engraftment based on human CD46 expression in PBMCs. (H) Editing measured using Sanger sequencing in PBMCs of secondary recipients at indicated weeks after transplantation. (I) Editing measured using Sanger sequencing at week 16 after transplantation in PBMCs, splenocytes, and BM cells. (J) Target base conversions and indel frequency measured using NGS. Week 16 BM MNCs samples from primary and secondary mice were used. K-N) Analyses of hemoglobin composition. Whole blood samples at week 16 after in vivo (n = 7) and ex vivo (n = 5) transduction were analyzed. Samples from untreated CD46/Townes mice (n = 3) were used as control. (K) Percentages of hemoglobin tetramers as measured using HPLC. Representative chromatograms are shown in supplemental Figure 12A. (L) Percentages of hemoglobin subunits as measured using mass spectrometry. (M) Representative chromatogram pattern showing the separation of β^A from β^S globin chains. The labeled percentages were calculated based on the peak areas. See supplemental Figure 12B for representative full spectrum chromatograms. (N) Separation of hemoglobin variants using isoelectric focusing electrophoresis. Each lane represents 1 mouse (ear tag number labeled) or AFSC controls. Bands in AFSC controls indicating 4 different hemoglobin variants are labeled. For B-D and F-J, each dot represents 1 animal. Data shown are mean ± standard deviation, wherever applicable. AFSC, hemoglobin A, F, S, and C controls.