Stable high-level expression of heterologous genes in vitro and in vivo bynoncytopathic DNA-based Kunjin virus replicon vectors

primary features of the flavivirus Kunjin (KUN) subgenomic replicons includ e continuous noncytopathic replication in host cell cytoplasm and the abili ty to be encapsidated into secreted virus-like particles (VLPs). Previously we reported preparation of RNA-based KUN replicon vectors and expression o f heterologous genes (HG) in cell culture after RNA transfection or after i nfection with recombinant KUN VLPs (A. N. Varnavski and A. A. Khromykh, Vir ology 255:366-375, 1999). In this study we describe the development of the next generation of KUN replicon vectors, which allow synthesis of replicon RNA in vivo from corresponding plasmid DNAs. These DNA-based vectors were a ble to direct stable expression of beta-galactosidase (beta-Gal) in several mammalian cell lines, and expression remained high (similar to 150 pg per cell) throughout cell passaging. The applicability of these vectors in vivo was demonstrated by beta-Gal expression in the mouse lung epithelium for a t least 8 weeks after intranasal inoculation and induction of anti-beta Gal antibody response after intramuscular inoculation of the beta-Gal-encoding KUN replicon DNA. The noncytopathic nature of DNA based KUN replicon vecto rs combined with high-level and stability of HG expression in a broad range of host cells should prove them to be useful in a variety of applications in vitro and in vivo.