An enhanced packaging system for helper-dependent herpes simplex virus vectors.
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.
Gene Therapy and Molecular Virology Group, The John P. Robarts Research Institute, London, Ontario, Canada N6A 5K8.
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Stavropoulos
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Strathdee
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D006224:Cricetinae; D004926:Escherichia coli; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics*; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D014709:Vero Cells; D014771:Virion; D019065:Virus Assembly