Evaluation of transduction with rAAV-HLP-codop-hFVIII-N6 in immunocompetent mice. (A) Kinetics of hFVIII:Ag expression following a single tail vein administration of 2 × 1013 vg/mouse of rAAV-HLP-codop-hFVIII-N6 pseudotyped with serotype 5 or 8 capsid in wild-type C57Bl/6 mice (N = 3/group). Shown are mean FVIII:Ag levels ± SD. (B, upper left) Southern blot of liver genomic DNA derived from mice (M1 and M2) transduced with rAAV8-HLP-codop-hFVIII-N6 was digested with Kpn I, a double cutter. Standards consist of rAAV-HLP-codop-hFVIII-N6 plasmid DNA spiked into naive mouse liver genomic DNA at the stipulated genome copies per cell concentration and then digested with Kpn I. (Upper right) Uncut DNA or DNA digested with a single cutter (Not I). HH, head-to-head and HT head-to-tail expected band size from proviral DNA in concatemeric configurations, respectively. (Bottom) A schematic showing the relative positions of Kpn I and Not I endonucleases in rAAV-HLP-codop-hFVIII-N6 when in a head to tail concatemeric configuration. (C) Western blot showing a single ∼210-kd band in the plasma of mice transduced with rAAV8-HLP-codop-hFVIII-N6, which is not present in naive mouse plasma. Positive control (+ve control) consisting of full-length recombinant hFVIII (Helixate) diluted in mouse plasma.
