Generation and characterization of E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII

The use of adenoviral vectors for gene therapy has been limited due to host immune responses directed toward the vector and/or transgene and vector to xicity. To decrease adenoviral vector immunogenicity and toxicity, we atten uated viral gene expression by eliminating El, E2a, E3, and E4 early genes from the adenoviral backbone. Two highly attenuated, fourth-generation (Av4 ) E1/E2a/E3/E4-deficient adenoviral vectors encoding human factor VIII (FVI II) under the control of a liver-specific albumin promoter were generated. One Av4 vector (Av4 Delta E4FVIII) was deficient in the entire E4 coding re gion and the second vector contained a deletion of the E4 region, except fo r open reading frame 3 (orf 3; Av4orf3FVIII). The Av4 vectors were compared to an E1/E2a/E3-deficient third-generation vector (Av3H8101) containing an analogous transgene expression cassette in vitro and in vivo following int ravenous administration in hemophiliac mice. In vitro transduction of Hep3B cells revealed at all three vectors expressed functional FVIII. However, t he Av4 Delta E4FVIII vector could not be scaled-up for in vivo studies. Bot h Av3H8101 and Av4orf3FVIII initially expressed similar levels of FVIII in hemophiliac mice. However, at 3 months, animals treated with the Av4orf3FVI II vector no longer expressed FVIII while Av3H8101-treated mice displayed p ersistent FVIII expression. Liver enzyme analyses of plasma samples reveale d that the Av4orf3FVIII vector was significantly less hepatotoxic than the Av3H8101 vector. These data demonstrate that further attenuation of the ade noviral vector backbone by removal of the majority of the E4 coding region significantly diminished vector toxicity; however, the duration of transgen e expression was reduced.