Figure 2.

LV.LCR-EFS.GAA-Transduced hCD34⁺ Cells Differentiate and Engraft in NSG Mice Similarly to Mock-Transduced Cells

Human CD34⁺ cells were transduced with LV.LCR-EFS.hGAAmCherry (LCRmCherry) or LV.EFS.eGFP (mock) at an MOI of 100 in the presence of LentiBOOST and protamine sulfate, and then kept in culture for a CFU assay or transplanted in NSG mice. (A) Number of differentiated colonies and (B) frequency distribution of total colony counts in mock- or LCRmCherry-transduced hCD34⁺ cells. Untreated group refers to healthy donor hCD34⁺ cells left unmanipulated as control group. NSG mice were sublethally irradiated to receive transduced hCD34⁺ cells (0.5 × 10⁶ cells/mouse; eight mice per group) via tail vein injection and culled 15 weeks later for analysis. Untreated refers to control NSG mice (total of four mice). (C and D) Representative fluorescence-activated cell sorting (FACS) plot of hCD45⁺ cells in bone marrow cells gated on live and lineage-negative (Lin-ve) cells (C) and their percentage distribution in untreated, mock, and LCRmCherry groups (D). (E–G) Representative FACS plot analysis of hCD34 hCD38 markers in bone marrow cells gated on Lin-ve hCD45⁺ cells (E) and the percentage distribution of hCD34⁺ (F) and hCD34⁺ hCD38⁻ (G) cells within Lin-ve hCD45⁺ cells per each group. (H) Percentage of hCD3⁺, hCD19⁺, and hCD33⁺ cells within hCD45⁺ bone marrow cells. (I and J) Percentage distribution of hCD45⁺ cells in PBMCs (I) and percentage of hCD3⁺, hCD19⁺, and hCD33⁺ cells in blood (J). (K and L) hGAAmCherry expression was detected in (K) bone marrow, PBMCs, and plasma by ELISA and (L) in hCD19⁺ and hCD33⁺ hCD45⁺ bone marrow cells by flow cytometry. The data are shown as means ± SD. ∗∗∗∗p < 0.0001, by by two-way ANOVA test, Sidak’s multiple comparison test.