Evaluation of expression and clinical effect after neonatal transduction of hemophilia B dogs.

Neonatal hemophilia B dogs were treated with gene therapy as described in the legend to Figure 6. The arrow for G08 at 4 months after transduction indicates a minor bleed that was treated with 2 doses of canine plasma. G18 died of pyelonephritis at 18 days after gene transfer, whereas G08 and G42 are still being followed. (A) Whole blood clotting time (WBCT). The whole blood clotting time is 8 to 13 minutes for normal dogs (gray shaded region), and longer than 60 minutes for hemophilia B dogs (hatched region). (B) aPTT coagulation assay. The range of aPTT for normal dogs from the same colony (average ± 2 standard deviations) is 11.6 to 12.6 seconds (gray shaded region). The range of aPTT for 5 untreated hemophilia B dogs from the same colony was 37.5 to 59.1 seconds (hatched region). (C) Percentage of normal cFIX activity. The percentage of normal cFIX activity in the plasma samples from the RV-treated dogs was determined by comparing the aPTT with a standard curve generated by means of mixtures of normal and cFIX-deficient dog plasma. The normal range (normal) of cFIX (60% to 160%) is indicated by the gray shaded region. Untreated hemophilia B dogs have less than 0.1% of normal activity (hatched region). (D) Percentage of normal cFIX antigen levels. The cFIX antigen levels were determined by immunoassay and compared with the level present in a pool of normal dog plasma from the Chapel Hill colony to determine the percentage of normal antigen levels. The normal range (normal) of cFIX is 60% to 160% (3 to 8 μg/mL) and is indicated by the gray shaded region. Untreated hemophilia B dogs (HB) have less than 0.02% of normal cFIX antigen levels, as indicated by the hatched region. (E) Anti-cFIX IgG antibody levels. Dogs were tested for anti-cFIX IgG antibodies as detailed in “Materials and methods.”