Evaluation of AAV FVIII variants in non-human primates. (A) hFVIII:Ag levels (solid black line; mean ± SD, left y-axis) and anti-hFVIII antibody titers (dashed gray line; relative units, right y-axis) as assessed by immunocapture assays over time in 4 2- to 4-year-old male monkeys following a single peripheral vein tail vein administration of either rAAV8-HLP-codop-hFVIII-N6 (N6) or rAAV8-HLP-codop-hFVIII-V3 (V3). Animals that developed neutralizing anti-hFVIII antibodies were treated with 4 monthly cycles of rituximab and cyclophosphamide shown by arrows. (B) Southern blot analysis showing molecular configuration of rAAV8-HLP-codop-hFVIII-N6 genome in macaque liver. Approximately 15 µg of DNA from each time point (1, 4, and 40 weeks after gene transfer) was electrophoresed uncut (uncut) or following digestion with Not I (single cutter) or Kpn I (double cutter). Shown are bands representing high-molecular-weight concatemers (HMWC) in the head-tail (HT) and head-to-head (HH) formation. (Bottom) A schematic of rAAV-HLP-codop-hFVIII-N6 in the HT configuration together with the cleavage sites for Kpn I and Not I.