HIGH-TITER BICISTRONIC RETROVIRAL VECTORS EMPLOYING FOOT-AND-MOUTH-DISEASE VIRUS INTERNAL RIBOSOME ENTRY SITE

Bicistronic retroviral vectors were constructed containing the foot-an d-mouth disease virus (FMDV) internal ribosome entry site (IRES) follo wed by the coding region of beta-galactosidase (beta-gal) or therapeut ic genes, with the selectable neomycin phosphotransferase gene under t he control of the viral long terminal repeat (LTR) promoter, LNFX, a v ector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine dea minase (ADA) and beta-gal. In transduced primary rat vascular smooth m uscle cells the cytokines were expressed at high levels, similar to th ose obtained from vectors employing the viral LTR promoter, LNFZ, a ve ctor encoding beta-gal, had a 10-fold increase in titer over that of L NPoZ, a comparable vector containing the poliovirus (Po) internal ribo some entry site, Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomy cin phosphotransferase (NPT), Overall, these vectors had titers betwee n 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth diseas e virus IRES provides high-titer bicistronic vectors with high-level t wo gene expression.