Target-site correction and sickle cell hemoglobin (HbS) to normal adult hemoglobin (HbA) conversion in HDAd-PE5max transduced mice after WU-106 + AMD3100 mobilization. (A) Target-site editing measured using Sanger sequencing. Editing at the sickle mutation site (T > A) and silent PAM site (G > A) are shown. Left panel: editing in PBMCs. Right panel: editing measured at week 16 after in vivo transduction in PBMCs, spleen, BM MNCs, pooled CFUs, and BM Lin–. Each dot represents an individual animal. (B) Target base conversions in BM MNCs (week 16), as measured by NGS. Each dot represents an individual animal. (C) Top alleles with frequencies >0.1% in mouse #4985, measured by NGS. (D) Allelic analysis of single Lin– cell–derived progenitor colonies. Editing was measured at day 11 after plating transduced Lin– cells. Shown are data from 3 mice with the indicated ear tag number. Twenty-four colonies from each mouse were analyzed. (E-F) Analyses of Hb composition. Whole blood samples at week 16 after in vivo transduction were analyzed. Samples from untreated CD46/Townes mice were used as a control. (E) Separation of Hb variants by IEF electrophoresis. Each lane represents 1 mouse (ear tag number labeled) or Hb AFSC controls. Bands in the AFSC controls, indicating 4 different Hb variants, are labeled. “ex vivo” indicates a control sample with near complete HbS to HbA conversion published previously.5 The sample was collected from a mouse transplanted with CD46/Townes Lin– cells ex vivo and transduced with HDAd-PE5max. The intensity of the bands was analyzed using ImageJ software and is summarized by the bar graph on the right. (F) Percentages of Hb variants measured by high-pressure liquid chromatography. For comparisons of multiple groups, 1-way and 2-way ANOVA with Bonferroni posttest for multiple comparisons was used. Statistical analysis was performed using GraphPad Prism version 10.0.3. ∗∗∗P ≤ .001, ∗∗P ≤ .01, ∗P ≤ .05.
