Long-term bleeding correction of B6/129-HA mice after FVIII gene transfer and effects of Tregs depletion after LV.pF8.FVIII delivery. (A) aPTT assay on plasma of LV.pF8.FVIII (n = 8) and LV.pF8.FVIII.co injected mice (n = 7) up to 68 weeks. (B) ELISA assay showing the absence of anti-FVIII antibodies after LV treatment. Bleeding assay (C) and bleeding time (D) demonstrating bleeding correction in all HA injected mice (P < .001), with a significant improvement of the HA phenotype mediated by FVIII.co form (P < .001). (E) hFVIII activity trend in plasma of B6/129-HA mice injected with LV.pF8.hFVIII (n = 10) and underwent Tregs depletion 11 weeks later (n = 5). Bethesda assay showing low titer inhibitors (F) and FVIII-specific isotypes IgG1, IgG2a, and IgG2b (G) in plasma of Tregs depleted mice. (H) Percentage of Tregs (CD4⁺ CD25⁺ Foxp3⁺) in PB of mice from both groups. *P ≤ .0001.