Construction and production of recombinant herpes simplex virus vectors

Virus vectors have been employed as gene transfer vehicles for various pre-clinical and clinical gene therapy applications. Replication-competent herpes simplex virus (HSV) vectors that replicate specifically in actively dividing glial tumor cells have been used in Phase I-II human trials in patients with glioblastoma multiforme (GBM), a fatal form of brain cancer. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, non-toxic, and capable of long-term transgene expression. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purification, and their small-scale production for use in cell culture experiments as well as studies in animals.