Generation of an adenovirus vector lacking E1, E2a, E3, and all of E4 except open reading frame 3

Toxicity and immunity associated with adenovirus backbone gene expression i s an important hurdle to overcome for successful gene therapy. Recent effor ts to improve adenovirus vectors for in vivo use have focused on the sequen tial deletion of essential early genes. Adenovirus vectors have been constr ucted with the E1 gene deleted and with this deletion in combination with a n E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lack ing E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressin g a beta-galactosidase reporter gene. This vector was generated by transfec tion of a plasmid carrying the full-length vector sequence into A30.S8 cell s that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57B L/6 mice that the Av4orf3nBg vector effected gene transfer with an efficien cy comparable to that of the Av3nBg (wild-type E4) vector but that the form er exhibited a higher level of beta-galactosidase expression. This observat ion suggests that E4 ORF3 alone is able to enhance RNA levels from the beta -galactosidase gene when the Rous sarcoma virus promoter is used to drive t ransgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected w ith the Av3RBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion backg round may be beneficial in decreasing immunogenicity and improving safety a nd toxicity profiles, as well as increasing transgene capacity and expressi on for liver-directed gene therapy.