A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus.
Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising tools for gene therapy. Since these vectors are deleted of all adenoviral genes, they require a helper adenovirus for their propagation. The contamination of the vector preparation by the helper limits the utility of currently existing FD-AdVs in gene therapy applications. We have developed an alternative system for the propagation of FD-AdVs, in which the adenoviral genes essential for replication and packaging of the vector are delivered into producer cells by a baculovirus-adenovirus hybrid. A hybrid baculovirus Bac-B4 was constructed to carry a Cre recombinase-excisable copy of the packaging-deficient adenovirus genome. Although the total size of the DNA insert in Bac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with titers of 5 x 10(8)p.f.u./ml before concentration. Transfection of 293-Cre cells with lacZ-expressing FD-AdV plasmid DNA followed by infection by Bac-B4 at a MOI of 2000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent passaging of the obtained FD-AdV using Bac-B4 as a helper resulted in approximately 100-fold increases of the vector titer at each passage. This resulting vector was completely free of helper virus and was able to transduce cultured 293 cells. However, scaling-up of FD-AdV production was prevented by the eventual emergence of replication-competent adenovirus (RCA). Experiments are underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replication-competent adenovirus (RCA) during vector production. This baculovirus-based system will be a very useful alternative to current methods for the production of FD-AdVs.
Cheshenko
N
N
Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
Krougliak
N
N
Eisensmith
R C
RC
Krougliak
V A
VA
D000256:Adenoviridae / Q000235:genetics*; D016367:Baculoviridae / Q000235:genetics*; D015139:Blotting, Southern; D002460:Cell Line; D017353:Gene Deletion; D015870:Gene Expression; D005814:Genes, Viral; D005818:Genetic Engineering; D005822:Genetic Vectors; D006801:Humans; D014161:Transduction, Genetic / Q000379:methods; D001616:beta-Galactosidase / Q000235:genetics
D001616:beta-Galactosidase