Significant levels of FVIII:Ag in plasma of hemophilia A dog treated with gene delivery. Hemophilia dog #3 weighed 9 kg and was 2 years old when 3.28 × 109 autologous cBOECs that expressed 1.5 U/mL per 106 cells over 24 hours in vitro were implanted into 13 sites. Prior to receiving the genetically modified cells, 8 infusions of recombinant FVIII were administered by intravenous injections. This dog received 3 infusions of recombinant FVIII (black arrows) on the day of the treatment. An additional 2 infusions of FVIII were given on days 42 and 59 after gene delivery. A second gene delivery treatment of 2 × 109 autologous FVIII-expressing cBOECs was implanted into 11 sites 63 days after the first treatment. Plasma samples were taken at the indicated times. (A) FVIII activity (FVIII:C) and FVIII antigen (FVIII:Ag) levels were measured. Normal canine plasma from 8 healthy dogs was pooled and used to generate the standard curve. Normal pooled plasma was arbitrarily set at 100%. Plasma samples from 13 untreated hemophilia A dogs showed no detectable FVIII:Ag. (B) Levels of inhibitory antibodies were measured by a Bethesda assay, and anti-FVIII IgG1 and IgG2 antibodies were measured by ELISA.
