Specific activity of F.IX-WT, F.IX-K5A/V10K, and F.IX-R338A following intramuscular injection of AAV vectors to C57Bl/6 hemophilia B/CD4 knock-out mice. (A) F.IX activity is determined in one-stage activated partial thromboplastin assay in mice plasma, and F.IX antigen levels are determined by ELISA. Animals were injected at 6 intramuscular sites in the hind limbs with AAV-F.IX-WT (▦;n = 15) or -R338A (; n = 8) at doses of 4 × 10¹² vg/kg or 8 × 10¹² vg/kg. AAV-K5A/V10K (▵) was injected at doses of 2 × 10¹¹ vg/kg (n = 4), 1.2 × 10¹² vg/kg (n = 4), or 4 × 10¹² vg/kg (n = 3). Plasma samples were collected at several time points after vector administration, and results from individual animals are indicated. (B) Factor IX expression in murine tissues harvested at week 18 following injection of an AAV vector under the control of the CMV enhancer/promoter. Shown are RT-PCR products for human factor IX sequences and for the murine hypoxanthine phosphoribosyltransferase (HPRT) as housekeeping gene. Gastrointestinal (GI) tract includes tissues from esophagus, stomach, and intestines. Densitometric analysis of RT-PCR shows that the majority of F.IX expression is derived from skeletal muscle and only a small fraction from the liver.