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Journal of Virology, December 2000, p. 11296-11303, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Episomal Segregation of the Adenovirus Enhancer Sequence by Conditional Genome Rearrangement Abrogates Late Viral Gene Expression

Xinzhong Wang, Weike Zeng, Masahiro Murakawa, Mason W. Freeman, and Brian Seed*

Nessel Gene Therapy Center and Departments of Medicine and Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts

Received 31 May 2000/Accepted 30 August 2000

We have constructed a recombinant adenovirus gene delivery system that is capable of undergoing growth phase-dependent site-specific recombination. When propagated in 293 producer cells, the vector retains its linear double-stranded form and can be propagated to high titer and purified by conventional procedures. Upon introduction into target cells, the viral chromosome undergoes cyclization to generate an autonomously replicating circular episome and a detached linear fragment. The viral enhancer and reporter gene segregate with the circular episome, which contains no adenovirus open reading frames. The effect of rearrangement of adenovirus gene expression was assessed by quantitative reverse transcription-PCR measurement of the abundance of transcripts encoding the tripartite leader sequence (TPL) of the major late promoter. Whereas nonrearranging viruses produced approximately 104 TPL transcripts per 106 infecting genomes in the HepG2 liver cell line, no transcripts were detectable in the same cells infected with comparable levels of circularizing vector. Because no helper virus is required to propagate these vectors, the problems of recombination with and contamination by helper virus are eliminated. We also present an efficient and reliable method for generating recombinant adenoviruses.

* Corresponding author. Mailing address: Department of Molecular Biology, Wellman 9, Massachusetts General Hospital, Boston, MA 02114. Phone: (617) 726-5975. Fax: (617) 726-5962. E-mail: seed{at}molbio.mgh.harvard.edu.

Journal of Virology, December 2000, p. 11296-11303, Vol. 74, No. 23
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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