General strategy for constructing large HSV-1 plasmid vectors that co-express multiple genes

Herpes simplex vir us type 1 (HSV-1) plasmid vectors have a number of attra ctive features for gene transfer into neurons. In particular the large size of the HSV-1 genome suggests that HSV-1 vectors might be designed to co-ex press multiple genes. Here, we report a general strategy for constructing l arge HSV-1 plasmid vectors that co-express multiple genes. Each transcripti on unit is linked to an antibiotic resistance gene, and genetic selections are used to assemble large vectors. Using this strategy, we constructed lar ge (26 or 31 kb) HSV-1 vectors that contain two transcription units and two or three genes. These vectors were efficiently packaged into HSV-1 pat-tid es using a helper virus-free packaging system. The resulting vector stocks supported the expression of two or three genes in both cultured cells and t he rat brain. Potential applications of HSV-1 vectors that co-express multi ple genes are discussed.