Production of recombinant Herpesvirus saimiri-based vectors

Herpesvirus saimiri (HVS) is capable of infecting a range of human cell types with high efficiency. The viral genome persists as high-copy-number, circular, nonintegrated episomes that segregate to progeny upon cell division. This allows HVS-based vectors to transduce stably a dividing cell population and provide sustained transgene expression for an extended period of time both in vitro and in vivo. Moreover, the insertion of a bacterial artificial chromosome (BAC) cassette into the HVS genome simplifies the incorporation of large amounts of heterologous DNA for gene delivery. These properties offer characteristics similar to that of an artificial chromosome combined with an efficient delivery system. To insert and express a heterologous gene in an HVS-based vector, a recombinant virus must be constructed, as described in this protocol. An HVS-BAC is used to simplify and enhance the production of recombinant viruses. This requires a two-step process to insert the heterologous expression cassette first into the pHVS-Shuttle and then into the HVS-BAC.