Complementation of a human adenovirus early region 4 deletion mutant in 293 cells using adenovirus-polylysine-DNA complexes.
The E1 deleted adenoviral vectors are efficient at gene transfer to cells in culture or in animals. However, their use is limited because of an immune-mediated loss of transduced cells. This immune response is believed to result from low-level production of viral antigens from these vectors after gene transfer. The early region 4 (E4) of adenovirus produces a number of proteins that play an important role in adenoviral and host gene regulation during infection of mammalian cells. There is interest in developing E4 deficient adenovirus for gene therapy, especially in the context of developing a combined E1/E4 deleted vector. Towards this goal, a method by which to complement and propagate an E4 deficient adenovirus (dl 1014) in the E1 complementing 293 cell line, using a novel and simple rescue technique, has been developed. Purified adenovirus deficient in E4 gene expression (dl 1014) was conjugated to expression plasmids containing the E4-open reading frame 6 gene or complete E4 region to produce adenovirus-polylysine-DNA complexes that were used to transfect 293 cells. The derived virus obtained from this transfection did not replicate on 293 cells but did replicate on W162 cells (E4+) confirming that the virus was indeed deleted for E4. Viral yield was high ranging from 3 x 10(8) to 9 x 10(8) plaque forming units per 10(6) 293 cells. This method has general application to the production of new adenoviral mutants that will be useful for developing second generation adenoviral vectors.
Scaria
A
A
Department of Medicine, Markey Molecular Medicine Center, University of Washington, Seattle 98195, USA.
Curiel
D T
DT
Kay
M A
MA
D017504:Adenovirus E4 Proteins / Q000235:genetics*; D000260:Adenoviruses, Human / Q000235:genetics; D017353:Gene Deletion; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D011107:Polylysine / Q000235:genetics*; D011995:Recombination, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D017504:Adenovirus E4 Proteins; D011107:Polylysine