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

Natalia V. Cheshenko, N. Krougliak, R. C. Eisensmith, V. A. Krougliak

Research output: Contribution to journalArticle

83 Citations (Scopus)

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 × 108 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 ∼ 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.

Original languageEnglish (US)
Pages (from-to)846-854
Number of pages9
JournalGene Therapy
Volume8
Issue number11
DOIs
StatePublished - 2001
Externally publishedYes

Fingerprint

Adenoviridae
Baculoviridae
Helper Viruses
Product Packaging
Genetic Therapy
Sf9 Cells
Genetic Structures
DNA
Essential Genes
Transfection
Insects
Cultured Cells
Plasmids
Genome
Infection

Keywords

  • Baculovirus
  • Baculovirus-adenovirus hybrid
  • Fully deleted adenovirus vector
  • Gene therapy
  • Production

ASJC Scopus subject areas

  • Genetics

Cite this

A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. / Cheshenko, Natalia V.; Krougliak, N.; Eisensmith, R. C.; Krougliak, V. A.

In: Gene Therapy, Vol. 8, No. 11, 2001, p. 846-854.

Research output: Contribution to journalArticle

Cheshenko, Natalia V. ; Krougliak, N. ; Eisensmith, R. C. ; Krougliak, V. A. / A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus. In: Gene Therapy. 2001 ; Vol. 8, No. 11. pp. 846-854.
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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 × 108 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 ∼ 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.",
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