IO delivery of G-F8-LV produced persistent FVIII expression in human megakaryocytes and platelets. Eight weeks after the transplant of 1 × 10⁶ human CD34⁺ cells, humanized NBSGW mice were IO-injected either with PBS (20 μL per animal) or with G-F8-LV (5 × 10⁷ ifu per animal). Twelve weeks following IO injection, platelets were collected from control mice injected with PBS (n = 5) and mice injected with G-F8-LV (n = 7) following CL treatment and lysed using 0.5% CHAPS. FVIII concentrations in platelets were measured by flow cytometry and ELISA. (A) Representative flow cytometry data from control and G-F8-LV–treated mice. The flow panels were gated for hCD41a⁺FVIII⁺ cells. Left, PBS injected; right, G-F8-LV injected. (B) Summary plot of FVIII⁺ cells in hCD41a⁺ platelets by flow cytometry (PBS, n = 4; G-F8-LV, n = 7). (C) Summary plot of FVIII concentrations measured in the supernatant of lysed platelets by ELISA. The data are presented as means with standard deviation from 3 separate experiments. ∗P < .05 and ∗∗P < .01. (D) BM of control or G-F8-LV–treated mice were harvested, and hCD34⁺ cells were isolated using autoMACS. The cells were grown in either CC110 or MEG-supplemented SFEM II media for 9 days, and intracellular expression of FVIII was measured by flow cytometry.