A GENE-TRANSFER VECTOR-CELL LINE SYSTEM FOR COMPLETE FUNCTIONAL COMPLEMENTATION OF ADENOVIRUS EARLY REGIONS E1 AND E4

The improvements to adenovirus necessary for an optimal gene transfer vector include the removal of virus gene expression in transduced cell s, increased transgene capacity, complete replication incompetence, an d elimination of replication-competent virus that can be produced duri ng the growth of first generation adenovirus vectors. To achieve these aims, we have developed a vector-cell line system for complete functi onal complementation of both adenovirus early region 1 (E1) and E4. A library of cell lines that efficiently complement both E1 and E4 was c onstructed by transforming 293 cells with an inducible E4-ORF6 express ion cassette. These 293-ORF6 cell lines were used to construct and pro pagate viruses with E1 and E4 deleted. While the construction and prop agation of AdRSV beta gal.11 (an E1(-)/E4(-) vector engineered to cont ain a deletion of the entire E4 coding region) were possible in 293-OR F6 cells, the yield of purified virus was depressed approximately 30-f old compared with that of E1(-) vectors. The debilitation in AdRSV bet a gal.11 vector growth was found to correlate with reduced fiber prote in and mRNA accumulation. AdCFTR.11A, a modified E1(-)/E4(-) vector wi th a spacer sequence placed between late region 5 and the right invert ed terminal repeat, efficiently expressed fiber and grew with the same kinetic profile and virus yield as did E1(-) vectors. Moreover, purif ied AdCFTR.11A yields were equivalent to E1(-) vector levels. Since no overlapping sequences exist in the E4 regions of E1(-)/E4(-) vectors and 293-ORF6 cell lines, replication-competent virus cannot be generat ed by homologous recombination. In addition, these second-generation E 1(-)/E4(-) vectors have increased transgene capacity and have been ren dered virus replication incompetent outside of the new complementing c ell lines.