Gene Therapy
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June 2001, Volume 8, Number 11, Pages 846-854
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Research Article
A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus
N Cheshenko1, N Krougliak1, R C Eisensmith1,2 and V A Krougliak1

1Institute for Gene Therapy and Molecular Medicine, Mount Sinai School of Medicine, New York, NY, USA

2Department of Human Genetics, Mount Sinai School of Medicine, New York, NY, USA

Correspondence to: V A Krougliak, Institute for Gene Therapy and Molecular Medicine, Box 1496, Mount Sinai School of Medicine, One Gustave L Levy Place, New York, NY 10029, USA

Abstract

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 ´ 108p.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 ~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. Gene Therapy (2001) 8, 846-854.

Keywords

fully deleted adenovirus vector; baculovirus; gene therapy; baculovirus-adenovirus hybrid; production

Received 1 August 2000; accepted 6 February 2001
June 2001, Volume 8, Number 11, Pages 846-854
Table of contents    Previous  Abstract  Next   Full text  PDF