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 mediate expression of high levels of human FVIII and correct the bleeding defect in haemophiliac mice and dogs. While vector assessment in a non-human primate model would have a significant preclinical benefit, a haemophiliac non-human primate model is not available, and assays that distinguish human FVIII 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 carboxy 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 was biologically active in vivo. To distinguish the BDD-E protein from non-human primate FVIII, a sensitive immunoprecipitation/Western assay was developed that reproducibly detected 1 ng mL-1 of the epitope-tagged human FVIII in the presence of monkey plasma. These data demonstrate that the addition 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 primate studies.