FIGURE 2.

Targeting the hFIX cDNA into the albumin locus of neonatal FIX KO mice. (A) Experimental design. FIX KO neonatal mice were injected at postnatal day (P) 2 with 0.6E11 vg/mouse of AAV8-SaCas9 and 5.0E11 vg/mouse of AAV8-donor-hFIX. Bleeding was performed at 1, 2 months (M) and mice were sacrificed at 4 months. The liver was collected for molecular analysis; (B) hFIX plasma levels (ng/ml) at 1, 2, and 4 months of age in WT (n = 11), Het (n = 6) and FIX KO (n = 15) mice transduced with SaCas9 and donor-hFIX treated at post-natal (P) day 2. 5,000 ng/ml corresponds to the normal FIX plasma levels in healthy individuals in the human population; (C) Western blot analysis of plasma hFIX in treated FIX KO mouse plasma. Untreated WT and KO mice were used as negative controls, while human plasma was used as a positive control; (D) Tail-bleeding assay. The coagulation time was evaluated in neonatally-treated FIX KO mice (n = 5) and their wild-type littermates (n = 8). Data are shown as mean ± SEM and analyzed by one-way ANOVA with Tukey’s multiple comparison test.