A novel system for the production of fully deleted adenovirus vectors thatdoes not require helper adenovirus

Fully deleted adenovirus vectors (FD-AdVs) would appear to be promising too ls for gene therapy. Since these vectors are deleted of all adenoviral gene s, they require a helper adenovirus for their propagation. The contaminatio n of the Vector preparation by the helper limits the utility of currently e xisting FD-AdVs in gene therapy applications. We have developed an alternat ive system for the propagation of FD-AdVs, in which the adenoviral genes es sential for replication and packaging of the vector are delivered into prod ucer 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 B ac-B4 was 38 kb, the genetic structure of this recombinant baculovirus was stable. Bac-B4 gave high yields in Sf9 insect cells, with liters 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 2 000 p.f.u./ml resulted in rescue of the helper-free vector. Subsequent pass aging of the obtained FD-AdV using Bac-B4 as a helper resulted in similar t o 100-fold increases of the vector titer at each passage. This resulting Ve ctor 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 e ventual emergence of replication-competent adenovirus (RCA). Experiments ar e underway to optimize this system for the large-scale production of helper virus-free FD-AdVs and to minimize the possibility of generation of replic ation-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.