EFFICIENT DUAL TRANSCOMPLEMENTATION OF ADENOVIRUS E1 AND E4 REGIONS FROM A 293-DERIVED CELL-LINE EXPRESSING A MINIMAL E4 FUNCTIONAL UNIT

Transgene expression after the administration of recombinant adenoviru s with El deleted is constantly transient. It is admitted that ELA-sub stituting activities of cellular or viral origin allow viral antigen s ynthesis and trigger cytotoxic lymphocyte-mediated clearance of the re cipient cells. Our approach to solving this problem relies on the addi tional deletion of the E4 region from the vector backbone as this regi on upregulates viral gene expression at both transcriptional and postt ranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of tran scomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was c loned under the control of the dexamethasone-inducible mouse mammary t umor virus long terminal repeat. Following transfection into 293 cells , clone IGRP2 was retained and characterized as it can rescue the grow th defect of all E1(+) E4(-) adenoviral deletants tested, DNA and RNA analysis experiments verified that the mouse mammary tumor virus promo ter drives the expression of the ORF6+ORF7 unit and permits its bona f ide alternative splicing, generating ORF6/7 mRNA in addition to the OR F6-expressing primary transcript. Importantly, IGRP2 cells sustain cel l confluence for a period longer than that of 293 parental cells and a llow the plaque purification of E1(-) or E4(-) defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is de monstrated by the construction, plaque purification, and helper-free p ropagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packag ing cell line will be drastically impaired as only a limited segment o f E4 has been integrated.