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Journal of Virology, September 1998, p. 7137-7143, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

An Enhanced Packaging System for Helper-Dependent Herpes Simplex Virus Vectors

Tom A. Stavropoulos and Craig A. Strathdee*

Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, London, Ontario, Canada N6A 5K8, and Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada N6A 5C1

Received 27 March 1998/Accepted 9 June 1998

Helper-dependent herpes simplex virus (HSV) vectors (amplicons) show considerable promise to provide for long-term transduced-gene expression in most cell types. The current packaging system of choice for these vectors involves cotransfection with a set of five overlapping cosmids that encode the full HSV type 1 (HSV-1) helper virus genome from which the packaging (pac) elements have been deleted. Although both the helper virus and the HSV amplicon can replicate, only the latter is packaged into infectious viral particles. Since the titers obtained are too low for practical application, an enhanced second-generation packaging system was developed by modifying both the helper virus and the HSV amplicon vector. The helper virus was reverse engineered by using the original five cosmids to generate a single HSV-bacterial artificial chromosome (BAC) clone in Escherichia coli from which the pac elements were deleted to generate a replication-proficient but packaging-defective HSV-1 genome. The HSV amplicon was modified to contain the simian virus 40 origin of replication, which acts as an HSV-independent replicon to provide for the replicative expansion of the vector. The HSV amplicon is packaged into infectious particles by cotransfection with the HSV-BAC helper virus into the 293T cell line, and the resulting cell lysate is free of detectable helper virus contamination. The combination of both modifications to the original packaging system affords an eightfold increase in the packaged-vector yield.

* Corresponding author. Mailing address: Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, P.O. Box 5015, 100 Perth Dr., London, Ontario, Canada N6A 5K8. Phone: (519) 663-5777, ext. 4032. Fax: (519) 663-3789. E-mail: cas{at}rri.on.ca.

Journal of Virology, September 1998, p. 7137-7143, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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