IN-VIVO GENE DELIVERY AND EXPRESSION OF PHYSIOLOGICAL LEVELS OF FUNCTIONAL HUMAN FACTOR-VIII IN MICE

Hemophilia A is caused by blood coagulation factor VIII (FVIII) defici ency and is an attractive target for gene therapy. However, features o f FVIII physiology, such as the instability of the mRNA and protein, h ave provided obstacles to the design of a feasible strategy for the tr ansfer and expression of the human FVIII gene in vivo. We have constru cted a recombinant adenoviral vector, Av1ALH81, that contains the huma n FVIII cDNA from which the B-domain has been deleted (BDD FVIII) and extensively characterized this vector in vitro and in vivo. In vitro, HepG2, human hepatoma cells, transduced with AV1ALH81 secreted high le vels of biologically active human BDD FVIII measured by the Coatest bi oassay (>2,400 mU per 10(6) cells per 24 hr). Administration of Av1ALH 81 to mice, via tail vein, resulted in expression of human BDD FVIII i n the mouse plasma at levels averaging 307 +/- 93 ng/ml 1 week post-in jection, measured by a sensitive human FVIII-specific ELISA. Normal FV III levels in humans are 100-200 ng/ml, and therapeutic levels are as low as 10 ng/ml. Purification of the human FVIII from the mouse plasma , and subsequent Coatest analysis, revealed that the human FVIII produ ced in the mice was biologically active. In addition, the duration of FVIII expression in vivo was followed, and high-level FVIII expression was sustained over a period of several weeks. The finding that an ade noviral vector can mediate high-level expression of human FVIII in an animal model provides the basis for the development of gene therapy fo r hemophilia A.