Functional activity of cFIX in RV-treated mice.

(A) Standard curve for samples from normal or hemophilia B mice. The aPTT coagulation assays were performed with the use of plasma from hFIX-deficient human plasma, plasma from normal or hemophilia B mice, and varying amounts of plasma from normal and cFIX-deficient dogs as detailed in “Materials and methods.” The average time to clot ± SEM is plotted versus the amount, in microliters, of normal dog plasma added for assays using plasma from normal mice (normal; ●) or hemophilia B mice (HB; ○). (B) In vitro coagulation activity for plasma from RV-treated mice. Normal BALB/c mice whose expression levels are shown in Figure 2B were treated with RV after HGF administration as young adults (adult normal; ●). To determine the amount of functional cFIX in the plasma, an aPTT assay was performed as detailed in “Materials and methods” and compared with the times obtained with the standards in panel A that used normal mouse plasma. The percentage of normal functional activity was plotted versus the percentage of normal antigen levels from the same animal. The line represents values where the antigenic and functional activity is the same. The hemophilia B mice whose expression levels are shown in Figure 3A were treated with RV shortly after birth (neonatal HB; ○). An aPTT assay was performed as detailed in “Materials and methods” and compared with the standards in panel A that used plasma from hemophilia B mice. (C) In vivo hemostasis assay in hemophilia B mice after neonatal transduction. The hemophilia B 129 × C57BL/6 mice whose expression levels are shown in Figure 3A were transduced with RV as newborns. Tail clip was performed at 4 months after transduction, and the percentage of the animals that achieved hemostasis within 15 minutes was determined. Tail clip was also performed on age-matched normal C57BL/6 and untreated hemophilia B 129 × C57BL/6 mice. The number of animals (N) in each group is indicated.