ELIMINATION OF BOTH E1 AND E2A FROM ADENOVIRUS VECTORS FURTHER IMPROVES PROSPECTS FOR IN-VIVO HUMAN GENE-THERAPY

A novel recombinant adenovirus vector, Av3nBg, was constructed with de letions in adenovirus El, E2a, and E3 regions and expressing a beta-ga lactosidase reporter gene, Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the ad enovirus El and E2a region genes inducibly expressed from separate glu cocorticoid-responsive promoters. Av3nBg demonstrated gene transfer an d expression comparable to that of Av1nBg, a first-generation adenovir us vector with deletions in El and E3, Several lines of evidence sugge st that this vector is significantly more attenuated than El and E3 de letion vectors, Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labe ling demonstrated no detectable de novo hexon protein synthesis for Av 3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell, Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions, In contrast, both Av1nBg and Av 2Lu vectors showed ed DNA replication and hexon protein synthesis at m ultiplicities of infection of 500 PFU per cell, Av2Lu has a deletion i n El and also carries a temperature-sensitive mutation in E2a, Thus, m olecular characterization has demonstrated that the Av3nBg vector is i mproved with respect to the potential for vector DNA replication and h exon protein expression compared with both first-generation (Av1nBg) a nd second-generation (Av2Lu) adenoviral vectors, These observations ma y have important implications for potential use of adenovirus vectors in human gene therapy.