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Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers

Gene Therapy volume 8, pages 111–120 (2001)Cite this article

Abstract

Recently developed helper virus-free methods of herpes simplex virus (HSV) amplicon vector packaging provide stocks that are virtually devoid of the cytotoxic component normally associated with traditional helper virus-based packaging methods. These approaches involve cotransfection of amplicon plasmid DNA with either a five-cosmid set or a bacterial artificial chromosome (BAC) that contains the HSV genome without its cognate pac signals. Helper virus-free amplicon packaging produces low-titer stocks (<105 expressing particles/ml) that exhibit a high frequency of pseudotransduction. In an effort to enhance amplicon titers, we introduced in trans a genomic copy of the virion host shutoff (vhs) protein-encoding gene UL41 into both cosmid- and BAC-based packaging strategies. Cotransfection of this plasmid with the amplicon and packaging reagents results in a 10-fold higher amplicon titer, and stocks that do not exhibit the pseudotransduction phenomenon. To further enhance packaging efficiency, the HSV transcriptional activator VP16 was introduced into packaging cells 1 day before the packaging components. Pre-loading of packaging cells with VP16 led to an additional enhancement of amplicon titers, an effect that did not occur in the absence of vhs. Increased helper virus-free amplicon titers resulting from these modifications will make in vivo transduction experiments more feasible.

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Acknowledgements

The authors thank Chris Chadwick for technical assistance in performance of histology and cell counting. An AFAR Research Grant to WJB and NIH R01-NS36420A to HJF supported this work.

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Authors and Affiliations

  1. Department of Neurology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    WJ Bowers, AI Brooks & HJ Federoff

  2. Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    WJ Bowers, DF Howard, AI Brooks & HJ Federoff

  3. Center for Aging and Developmental Biology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA

    MW Halterman & HJ Federoff

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  1. WJ Bowers
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Bowers, W., Howard, D., Brooks, A. et al. Expression of vhs and VP16 during HSV-1 helper virus-free amplicon packaging enhances titers. Gene Ther 8, 111–120 (2001). https://doi.org/10.1038/sj.gt.3301340

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