In vivo evaluation of a novel epitope-tagged human factor VIII-encoding adenoviral vector

Haemophilia A is caused by a deficiency in coagulation factor VIII (FVIII) and is an attractive target for gene therapy. Adenoviral vectors encoding a human B-domain deleted (BDD) FVIII cDNA have been shown previously to medi ate expression of high levels of human FVIII and correct the bleeding defec t in haemophiliac mice and dogs. While vector assessment in a non-human pri mate model would have a significant preclinical benefit, a haemophiliac non -human primate model is not available, and assays that distinguish human FV III from monkey FVIII have not been developed successfully. As a first step to enable vector evaluation in non-human primates, we have constructed an epitope-tagged FVIII molecule by the addition of 16 amino-acids to the carb oxy terminus of the BDD protein (BDD-E). Following vector administration to normal mice, therapeutic levels of BDD-E FVIII were expressed for at least 20 weeks. Treatment of haemophiliac mice revealed that the BDD-E protein w as biologically active in vivo. To distinguish the BDD-E protein from non-h uman primate FVIII, a sensitive immunoprecipitation/Western assay was devel oped that reproducibly detected 1 ng mL(-1) of the epitope-tagged human FVI II in the presence of monkey plasma. These data demonstrate that the additi on of an epitope tag had no effect on FVIII function or immunogenicity, and suggest that the BDD-E vector will be an effective reagent for non-human p rimate studies.