Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted

Heshan Zhou, Wanda O'Neal, Nuria Morral, Arthur L. Beaudet

Research output: Contribution to journalArticle

79 Citations (Scopus)

Abstract

Although adenovirus vectors offer many advantages, it would be desirable to develop vectors with improved expression and decreased toxicity. Toward this objective, an adenovirus vector system with deletion of both the E1 and E2a regions was developed. A 5.9-kb fragment of the adenovirus type 5 (Ad5) genome containing the E2a gene and its early and late promoters was transfected into 293 cells. A complementing cell line, designated 293-C2, expressed the E2a mRNA and protein and was found to complement the defect in Ad5 viruses with temperature-sensitive or deletion mutations in E2a. A deletion of 1.3 kb removing codons 40 to 471 of the 529 amino acids of E2a was introduced into plasmids for preparation of viruses and vectors. An AdS virus with disruption of the E1 gene and deletion of E2a grew on 293-C2 cells but not on 293 cells. Vectors with E1 and E2a deleted expressing Escherichia coli β-galactosidase or human α1-antitrypsin were prepared and expressed the reporter genes after intravenous injection into mice. This vector system retains sequences in common between the complementing cell line and the vectors, including 3.4 kb upstream and 1.1 kb downstream of the deletion. These vectors have potential advantages of increased capacity for insertion of transgene sequences, elimination of expression of E2a, and possibly reduction in expression of other viral proteins. Although the titers of the vectors with deleted are about 10- to 30-fold below those of vectors with E2a wild-type regions, the former vectors are suitable for detailed studies with animals to evaluate the effects on host immune responses, on duration of expression, and on safety.

Original languageEnglish (US)
Pages (from-to)7030-7038
Number of pages9
JournalJournal of Virology
Volume70
Issue number10
StatePublished - Oct 1996
Externally publishedYes

Fingerprint

Adenoviridae
cell lines
Cell Line
Viruses
viruses
Galactosidases
galactosidases
vector competence
Sequence Deletion
Insertional Mutagenesis
viral proteins
Gene Deletion
Viral Proteins
cells
Transgenes
Reporter Genes
reporter genes
intravenous injection
codons
Codon

ASJC Scopus subject areas

  • Immunology

Cite this

Development of a complementing cell line and a system for construction of adenovirus vectors with E1 and E2a deleted. / Zhou, Heshan; O'Neal, Wanda; Morral, Nuria; Beaudet, Arthur L.

In: Journal of Virology, Vol. 70, No. 10, 10.1996, p. 7030-7038.

Research output: Contribution to journalArticle

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