Development of Cell Lines Capable of Complementing E1, E4, and Protein IX Defective Adenovirus Type 5 Mutants

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Abstract
The cloning capacity of currently available E1- and E3-deleted adenovirus (Ad) vectors does not exceed 8 kb. To increase capacity and improve vector safety further, we have explored the possibility that Early Region 4 (E4) and the gene encoding protein IX (pIX) might also be deleted. To generate cell lines expressing sufficient levels of E4 and pIX proteins in trans in addition to E1-encoded proteins to complement mutations in these genes, we transformed 293 cells with constructs containing the E4 transcription unit and pIX coding sequences under the control of inducible mouse mammary tumor virus (MMTV) and metallothionein promoters, respectively. We obtained two lines, VK2-20 and VK10-9, that express both E4 and pIX proteins as well as E1. The lines could be efficiently transfected with DNA, and allowed the rescue and propagation of an adenovirus recombinant, Ad5dlE3,4, containing a 2.7-kb E3 deletion and a 2.8-kb E4 deletion in addition to an insertion of plasmid DNA sequences in E1A. Because the E4 sequences within VK2-20 and VK10-9 cells do not overlap with the DNA sequence of Ad5dlE3, E4, the probability of regeneration of the wild-type E4 during virus propagation should be very low. Using the cell lines described in this study, it should be possible to generate Ad vectors lacking E1, pIX, E3, and E4. This would not only increase capacity over that of currently available vectors (to ~11 kb) but would also result in more severely attenuated vectors than those with deletions only of E1 or of E1 and E3 and, hence, safer for use in gene therapy protocols. Adenovirus vectors are among the most promising systems for efficient gene transfer into mammalian cells. Current adenovirus vectors, with E1 and E3 deleted, have a cloning capacity of approximately 8 kb. To accommodate larger genes and to improve vector safety, it would be useful if E4 and pIX could also be deleted from the adenovirus genome. Because these genes are essential for virus growth, we constructed two cell lines, VK2-20 and VK10-9, that complement pIX and E4 mutants as well as E1-defective mutants. Using these cell lines, an adenovirus recombinant, Ad5dlE3, E4, was constructed that lacks the entire E3 and E4 regions and contains an insert of plasmid DNA in E1.

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